Inhibition of TRAIL-induced apoptosis by IL-8 is mediated by the p38-MAPK pathway in OVCAR3 cells.

INTRODUCTION: TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of the TNF superfamily of cell death inducing ligands. Interestingly, while malignant cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells do not appear to be sensitive to this ligand, making it a desirable therapeutic compound against many cancers, including many ovarian carcinomas. Interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be at significantly higher level in the ascites from patients with ovarian cancer. We have previously demonstrated a role for IL-8 in blocking TRAIL's ability to induce apoptosis in the ovarian cancer cell line, OVCAR3, possibly by repressing the DR4 TRAIL receptor expression and blocking caspase-8 cleavage. In addition, we showed a member of the mitogen-activated protein kinase (MAPK) superfamily, p38gamma, is among the genes regulated in OVCAR3 cells by TRAIL and IL-8. The present study further investigates involvement of the p38 MAPK pathway in IL-8's ability to block TRAIL-induced apoptosis in the ovarian surface epithelial cancer cell line, OVCAR3. RESULTS: In this study we demonstrate that p38gamma as well as p38alpha play a significant role in IL-8's ability to block TRAIL-induced apoptosis. Through array analysis, as well as confirmation with other methods, we detected regulation of p38gamma and p38alpha following treatment of the cancer cell line with IL-8 or TRAIL. We also tested two other isoforms of p38 MAPK, p38beta and p38delta, but did not find significant regulation by IL-8 or TRAIL. We also examined activation of the p38 MAPK pathway, up-stream as well as down-stream, and noticed activation of the pathway following treatment with TRAIL and decreased activity when IL-8 was introduced. With the use of specific inhibitors, we were able to further confirm the role of this pathway in TRAIL-induced apoptosis, and IL-8's ability to block this apoptosis, in ovarian cancer cell lines. CONCLUSION: Taken together, these results further solidify the role of IL-8 in blocking the TRAIL-induced apoptosis in these ovarian carcinoma cells and provide new molecular insight into this potentially important therapeutic target.

Glucocorticoid-induced apoptosis in lymphocytes.

Although the effects of glucocorticoids on lymphocytes have been scrutinized for years, researchers have yet to determine how these hormones induce apoptosis in susceptible cells. Compelling evidence indicates that DNA binding of the GR and subsequent transcriptional regulation of specific genes is required for this process. However, it is not clear whether the activation or repression of responsive genes is essential and more importantly, which of these genes, if any, are responsible for the induction of apoptosis. This review will focus on how glucocorticoid-induced apoptosisin lymphocytes is mediated by the glucocorticoid receptor, including a discussion of GR structure, function, and recent data implicating its role in the apoptotic process.

Inhibition of mineralocorticoid-mediated transcription by NF-kappaB.

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor that regulates the expression of multiple inflammatory and immune response genes and plays a critical role in host defense and in chronic inflammatory diseases. The mineralocorticoid receptor (MR) belongs to the steroid/thyroid hormone receptor super-family of ligand-induced transcription factors. We demonstrate a dose-dependent, mutual transcriptional antagonism between NF-kappaB and MR in transient transfection experiments. We also show that the antagonism is limited to the p65 subunit of NF-kappaB heterodimer but not p50. Transient cotransfection experiments with MR deletion constructs reveal the necessity of various N-terminal MR domains for this phenomenon. Inhibition of NF-kappaB by IkappaB relieves the repression of NF-kappaB function by MR. These data suggest that the p65 subunit of NF-kappaB interacts with MR indirectly and transrepresses MR activation.

Synthesis of a bacteriophage MB78 late protein by novel ribosomal frameshifting.

MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.

Phosphorylation of the insect immunophilin FKBP46 by the Spodoptera frugiperda homolog of casein kinase II.

Immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes, that exhibit peptidylprolyl isomerase (PPIase) activity. Members of this family bind to immunosuppressive drugs and on this basis are divided into two classes: FKBPs bind to FK506 and rapamycin, while cyclophilins bind to cyclosporin A. In this paper, we report on insect immunophilin FKBP46 and its associated kinase. The insect FKBP46 belongs to the high-molecular-weight immunophilins and shares many characteristic features with its mammalian counterparts, but its functional role remains unclear. Here, we show that FKBP46 is phosphorylated by a protein kinase present in the nucleus of both insect Spodoptera frugiperda (Sf9) and human Jurkat cells. This protein kinase is immunoreactive with polyclonal antiserum raised against Drosophila melanogaster casein kinase II (CKII). We have cloned, overexpressed and characterized a new member of the CKII family derived from Spodoptera frugiperda cells. Recombinant Sf9 CKII alpha subunit shares 75% identity to human, chicken and Drosophila melanogaster homologs, whereas the Sf9 CKII beta subunit is 77% identical to rat, chicken and human. Moreover, we demonstrate that the insect immunophilin FKBP46 can be phosphorylated by human and Sf9 casein kinase II. Finally, we show that FKBP46 interacts with DNA, and this interaction is not prevented by phosphorylation.

Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils.

IL-5 is a potent eosinophil viability-enhancing factor that has been strongly implicated in the pathogenesis of IgE-mediated inflammation in vivo. Recently published data have suggested that IL-5 (and related cytokines) may act by altering the expression of the anti-apoptotic regulator Bcl-2 or its homologues, but this is controversial. The behaviour of the recently described pro-apoptotic cysteine proteases (caspases) in eosinophils after IL-5 treatment has not been explored. We examined the effect of IL-5 on the expression of four major Bcl-2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti-apoptotic Mcl-1 and pro-apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl-x, Bcl-2. IL-5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl-2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL-5 completely blocked caspase processing in spontaneous and dexamethasone-induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL-5 acts by altering the balance of anti-apoptotic and pro-apoptotic Bcl-2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.

Transcriptional regulation of the human Na/K ATPase via the human mineralocorticoid receptor.

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) are members of the steroid/thyroid hormone receptor superfamily of ligand inducible transcription factors and have been shown to bind the glucocorticoid response element (GRE). Sodium-potassium ATPase (Na/KATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase alpha1 subunit and beta1 subunit genes transcriptionally. However, the mechanisms of corticosteroid regulation of mammalian Na/K ATPase subunit gene expression are not known. In this investigation, we report for the first time that cell lines (T-84 and 293) express endogenous MR by RT-PCR message expression. However, the protein product was not expressed as determined by western blot analyses. In transactivation studies of MR with GRE31, we detected MR expression at low concentrations of aldosterone. We also performed Northern blot and nuclear run-off transcription assays to further confirm that the regulation is transcriptional. We conclude that the transcriptional regulation of the human Na/K ATPase alpha1 and beta1 subunits by aldosterone occurs via the involvement of the MR.

The N-terminal domain of the mineralocorticoid receptor modulates both mineralocorticoid receptor- and glucocorticoid receptor-mediated transactivation from Na/K ATPase beta1 target gene promoter.

Mineralocorticoid and glucocorticoid hormones activate the expression of the Na/K ATPase beta1 through direct binding of the mineralocorticoid receptor (MR) and glucocorticoid receptors (GR) to a mineralocorticoid- and glucocorticoid-responsive element in the beta1 promoter region, but activation of the beta1 promoter is inhibited by coexpression of both receptors. Here, using a series of mutated and chimeric receptors, we show that the N-terminal region of MR mediates an inhibitory effect on MR and GR activation from the beta1 promoter, in CV-1 cells. Deletion of the N-terminal region of MR (1-603) enhanced MR activation four-fold. Activation by chimeric MR, in which the N-terminus of GR replaces the N-terminal region of MR, was threefold that of wild-type MR. In addition, whereas coexpression of wild-type MR and GR was inhibitory, coexpression of chimeric MR and wild-type GR was nearly equal to that of MR. By contrast, mutated GR lacking its N-terminal region (1-420) was less efficient than the wild type in activating this promoter. These results demonstrate that the N-terminal domains of MR and GR have opposite transactivation properties and that MR region 1-603 is indeed inhibitory for both MR- and GR-mediated regulation of the Na/K ATPase beta1 gene promoter.

Identification of a mineralocorticoid/glucocorticoid response element in the human Na/K ATPase alpha1 gene promoter.

Sodium-potassium ATPase (Na/K ATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) have been shown to bind the glucocorticoid response element (GRE); however, a specific element responsible for the activation of the MR is not known. Sequence analysis of the putative regulatory region of the Na/K ATPase alpha1 gene revealed the presence of a hormone response element that allows the MR to interact with it, at least as well as if not better than the GR. This response element is designated MRE/GRE. In this investigation, we demonstrated the MR and GR induced gene expression in COS-1 cells by cotransfecting with respective expression plasmids (RshMR and RshGR) along with a luciferase reporter. The synthetic MRE/GRE linked to a neutral promoter was activated by MR (6-fold); however, the GR induced a lower level of expression (3.8-fold), suggesting that the element may be preferably MR responsive. Mutations in the synthetic MRE/GRE could not induce the expression with MR, whereas GR had a small effect. Electrophoretic mobility shift analyses demonstrated a direct interaction of MR and GR with the MRE/GRE that was supershifted by an antiMR antibody and the complex was partially cleared by an antiGR antibody, respectively, whereas nonimmune serum had no effect. Footprinting analyses of the promoter region showed that a portion of the DNA containing this element is protected by recombinant MR and GR. Thus these data confirm that this MRE/GRE interacts with both MR and GR but interaction with receptors may be more MR-responsive than response elements previously described.

Upregulation of mineralocorticoid- and glucocorticoid-receptor gene expression by Sp-I.

The mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) belong to the steroid/thyroid hormone superfamily of ligand-induced transcription factors. Both activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. To assess the role of the transcription factor Sp1 and the nuclear factor I (NF-I), in MR- and GR-mediated gene expression using the human Na/K ATPase beta1 full-length promoter, we have examined the functions of Sp-I and NF-I functions in two different cell lines, COS-1 and T-84. By transient transfections we have shown that Sp-I significantly enhances MR and GR expression, whereas NF-I had negligible effect. We propose that the transcriptional enhancement could be through a direct interaction physically between MR or GR with Sp1 that allows other factors to bind the responsive element resulting in synergistic upregulation of transcription.

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae.

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.

Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.

p21-activated protein kinase gamma-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant gamma-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [gamma-32P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (approximately 5 min) is observed before autophosphorylation and activity are detected. When gamma-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of gamma-PAK in the presence of Cdc42(GTPgammaS) or histone 4, both cleavage products contain phosphate and gamma-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of gamma-PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain.

11Beta-hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules.

Mineralocorticoid hormones regulate salt transport along the distal nephron by binding to intracellular receptors and activating gene transcription. Previous experiments showed that systemic aldosterone infusions stimulate thiazide-sensitive Na and Cl transport by distal convoluted tubule (DCT) cells; this effect could have been direct or secondary to systemic hormonal effects. Aldosterone target tissues express both mineralocorticoid receptors and the metabolic enzyme 11beta-hydroxysteroid dehydrogenase type 2. Mineralocorticoid receptors have been localized to the DCT in some experiments, but not in others. Expression of 11beta-hydroxysteroid dehydrogenase type 2 by DCT cells has not been investigated. The present experiments were designed to test the hypothesis that rat DCT cells are targets of aldosterone action. Patterns of mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase, thiazide-sensitive Na-Cl cotransporter, and Na/Ca exchanger expression along the distal tubule were examined. A polyclonal antibody was generated to localize the thiazide-sensitive Na-Cl cotransporter. Thiazide-sensitive Na-Cl cotransporter and 11beta-hydroxysteroid dehydrogenase expression were examined using both in situ hybridization and immunocytochemistry; Na/Ca exchanger and mineralocorticoid receptor expression were examined by immunocytochemistry. The results indicate that 11beta-hydroxysteroid dehydrogenase is expressed by DCT cells, as well as connecting tubule cells and principal cells of the collecting duct; expression levels are low near the junction with the thick ascending limb and rise near the transition to the connecting tubule. Mineralocorticoid receptors are expressed by DCT cells, as well as along the thick ascending limb, connecting tubule, and collecting duct. The results indicate that components of the mineralocorticoid receptor system are expressed by DCT cells, suggesting that these cells are targets of aldosterone action.

Regulation of the human Na/K-ATPase beta1 gene promoter by mineralocorticoid and glucocorticoid receptors.

Expression of the human Na/K-ATPase beta1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions -650 to -630, within the beta1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the beta1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase beta1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the beta1 promoter effectively down-regulates transcription.

Calpain activation is upstream of caspases in radiation-induced apoptosis.

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.

FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis.

We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.

Identification of novel glucocorticoid-response elements in human elastin promoter and demonstration of nucleotide sequence specificity of the receptor binding.

Glucocorticoids exert their action on gene expression through activation of cytoplasmic glucocorticoid receptors (GRs) that bind to glucocorticoid response elements (GREs). The consensus GRE consists of two half sites (underlined), AGAACANNNTGTTCT. We have recently cloned the entire human elastin gene. Nucleotide sequencing of the promoter region disclosed the presence of three putative GREs with the downstream half-site sequence TGTTCC that has homology with the consensus GRE, although the upstream half site showed no homology. To examine the functionality of these putative GREs in binding to the GRs, we performed gel mobility shift and supershift assays with synthetic oligomers containing the putative GREs and a recombinant GR protein, expressed in a baculovirus system. All three GREs identified in the elastin promoter bound the receptor. A chimeric oligonucleotide containing the upstream consensus GRE half site and the downstream elastin promoter GRE half site was capable of binding the receptor, and this binding could be competed with the elastin promoter GRE. Nonconservative substitution of single nucleotides (positions 1-6) in the elastin GRE indicated that mutations in the positions 1-3 and 6 had relatively little effect, but substitutions in positions 4 and 5 rendered the oligomer less effective in competing for the binding. These observations suggest that the downstream half site of GREs in the human elastin promoter is sufficient for receptor binding and certain nucleotides are critical for the efficient binding. The results also imply that the three GREs within the human elastin promoter are active and mediate the glucocorticoid-induced up-regulation of human elastin promoter activity.

Nucleotides and two functional states of hsp90.

Previous studies have demonstrated the ATP-dependent formation of a complex containing the heat shock protein hsp90, the unique hsp90 binding protein p23, and one of three high molecular weight immunophilins. In the present study, hsp90 and p23 are shown to form a complex that requires elevated temperature and ATP/Mg2+. Complex formation is strongly promoted by molybdate and by the nonionic detergent Nonidet P-40. ADP and the benzoquinone ansamycin, geldanamycin, are potent inhibitors of complex formation. The ATP-dependent process alters the state of hsp90, not p23, and influences the ability of hsp90 to bind to phenyl-Sepharose. Conversion of hsp90 to the ATP-bound state lowers its affinity for phenyl-Sepharose. These results show that hsp90 can exist in at least two functional states, one able to bind p23 and the other with a high affinity for hydrophobic resins. A model is presented where these states are dictated by the binding of either ATP or ADP.

CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP.

FADD/MORT1 is a death domain (DD)-containing adaptor/signaling molecule that interacts with the intracellular DD of FAS/APO-I (CD95) and tumor necrosis factor receptor 1 and the prodomain of caspase-8 (Mch5/MACH/FLICE). FADD engagement of caspase-8 presumably activates this caspase and leads to apoptosis. Another DD-containing adaptor/signaling molecule, CRADD, was identified and was shown to induce apoptosis. CRADD has a dual-domain structure similar to that of FADD. It has an NH2-terminal caspase homology domain that interacts with caspase-2 and a COOH-terminal DD that interacts with RIP. CRADD is constitutively expressed in many tissues and thus could play a role in regulating apoptosis in mammalian cells.

The unliganded mineralocorticoid receptor is associated with heat shock proteins 70 and 90 and the immunophilin FKBP-52.

The human mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily, which includes receptors for retinoic acid, vitamin D, and other steroids, such as the glucocorticoids (which bind the glucocorticoid receptor, GR). MR and GR, the corticosteroid receptors, share significant homology and are activated by steroid binding, resulting in a conformational change, nuclear translocation, and DNA binding. Despite these similarities with GR, the MR remains less well characterized. However, protein components known to be present in the unliganded GR are also likely to be components of the heteromeric MR complex. In the current study, we investigated whether or not hsp70, hsp90, and the immunophilin FKBP-52 are present in the nonsteroid-bound MR complex, because these proteins are known to be present in the unliganded GR complex. The unliganded MR complex was assembled in vitro using reticulocyte lysate and in vivo using the baculovirus overexpression system and Spodoptera frugiperda (Sf9) cells. Western blot analysis revealed the presence of hsp70, hsp90, and FKBP-52 in the unliganded complexes, but hsp90 and FKBP-52 were not detected following exposure to aldosterone. Electrophoretic mobility shift analysis demonstrated that DNA binding of MR occurred only after treatment with aldosterone. These studies indicate that proteins associated with the unliganded GR are also present in the unliganded MR complex, and that hsp90 and FKBP-52 dissociate prior to DNA binding in a manner similar to that described for GR. Finally, the stoichiometric analysis of the proteins present within the heteromeric MR complex suggests a divergence between this receptor and the GR.