Impaired transactivation of the glucocorticoid receptor cloned from the Guyanese squirrel monkey

Squirrel monkeys are among a diverse group of New World primates that demonstrate unusually high levels of circulating corticosteroids and glucocorticoid receptor (GR) insensitivity. Recent evidence suggests that overexpression of an immunophilin impairs dexamethasone binding to GR in the Bolivian squirrel monkey (Saimiri boliviensis). Here we describe the cloning, expression, and functional characterization of GR from the closely related Guyanese squirrel monkey (S. sciureus). The cloned Guyanese squirrel monkey GR (gsmGR) cDNA closely resembles human GR (hGR) cDNA, and yields a high affinity dexamethasone binding receptor when expressed in COS-1 cells. Transactivation analysis of hGR and gsmGR expressed in CV-1 cells and cultured squirrel monkey kidney (SMK) cells indicates that: (1) SMK cells elaborate a functional high activity GR from human GR cDNA; (2) gsmGR is an order of magnitude less efficient than hGR at transactivation in CV-1 and SMK cells; and (3) maximal transactivation by gsmGR is attenuated in both cell lines. Glucocorticoid resistance in S. sciureus is at least partly attributable to a naturally occurring mutation in the GR gene that results in impaired GR transactivation.     

Overexpression and characterization of the human mineralocorticoid receptor.

The full-length human renal mineralocorticoid receptor (hMR) has been overproduced in Spodoptera frugiperda (Sf9) insect cells using baculovirus-mediated expression. The overproduced hMR binds aldosterone with high affinity (Kd = 1.36 nM) and has high affinity for cortisol, cortexolone, and progesterone. Immunoprecipitation and immunoblot analysis of the recombinant hMR with MR-specific antibodies reveal three major protein bands with molecular masses of 115, 119, and 125 kDa. hMR isoforms show maximal accumulation at 48 h post-infection with the recombinant baculovirus. Maximal aldosterone binding was detected at 24 h rather than at 48 h post-infection, suggesting that the assembly of hMR monomers into the nonactivated steroid-binding receptor complexes and/or their stability deteriorates after 24 h post-infection. It is estimated by specific aldosterone binding that 1.2 x 10(6) hMR molecules are expressed per Sf9 cell (equivalent to 7 pmol/mg of cytosolic protein) at 24 h post-infection. 5-Fold more receptor molecules/cell are expressed but not detected by steroid binding at 48 h post-infection as determined by immunoblot analysis. Using the MR-specific H10E anti-idiotypic monoclonal antibody, immunoprecipitation of cytosol from recombinant baculovirus-infected Sf9 cells pulse-labeled with 32Pi demonstrated for the first time that the recombinant hMR is highly phosphorylated. The hMR is expressed as 9-10 S oligomeric complexes (Stokes radii approximately 67-85 A) that are slightly heavier than the unactivated glucocorticoid receptor and can be converted to smaller 4 S receptor monomers (Stokes radii approximately 25-55 A) by elevated temperature, pH, and ionic strength. Unlike the glucocorticoid receptor, the oligomeric hMR complex can bind DNA-cellulose without prior activation. Finally, indirect immunofluorescence demonstrated that the hMR is expressed primarily as a cytoplasmic protein that can be induced to translocate to the nucleus upon treatment with hormone.     

The role of hsp90 in heme-dependent activation of apo-neuronal nitric-oxide synthase

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S., Bender, A. T., Kanelakis, K. C., Murphy, P. J. M., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 278, 15465-15468). In this work, we examine heme binding by apo-nNOS to form the active enzyme in a cell-free system. We show that cytosol from Sf9 cells facilitates heme-dependent apo-nNOS activation by promoting functional heme insertion into the enzyme. Sf9 cytosol also converts the glucocorticoid receptor (GR) to a state where the hydrophobic ligand binding cleft is open to access by steroid. Both cell-free heme activation of purified nNOS and activation of steroid binding activity of the immunopurified GR are inhibited by radicicol treatment of Sf9 cells prior to cytosol preparation, and addition of purified hsp90 to cytosol partially overcomes this inhibition. Although there is an hsp90-dependent machinery in Sf9 cytosol that facilitates heme binding by apo-nNOS, it is clearly different from the machinery that facilitates steroid binding by the GR. hsp90 regulation of apo-nNOS heme activation is very dynamic and requires higher concentrations of radicicol for its inhibition, whereas GR steroid binding is determined by assembly of stable GR.hsp90 heterocomplexes that are formed by a purified five-chaperone machinery that does not activate apo-nNOS.     

Complex human glucocorticoid receptor dim mutations define glucocorticoid induced apoptotic resistance in bone cells

A mutation in the D-loop of the second zinc finger of the DNA-binding domain of the human glucocorticoid receptor (hGR), A458T (GR(dim)), has been suggested to be essential for dimerization and DNA binding of the GR, and genetically altered GR(dim) mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GR(dim) mice were reported to be resistant to glucocorticoid-induced apoptosis. To further evaluate the dim mutations in glucocorticoid-induced apoptosis, we stably expressed either the hGR(dim) (A458T) or the hGR(dim4) (A458T, R460D, D462C, and N454D) mutant receptors in human osteosarcoma (U-2 OS) cells that are devoid of hGR and unresponsive to glucocorticoids. We analyzed these cell lines by comparison with a stable expression hGRα U-2 OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGR(dim) mutation had diminished steroid responsiveness and cells carrying the hGR(dim4) mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor κB repression was unaffected by either mutation. Interestingly, both the hGR(dim) and hGR(dim4) receptors readily formed dimers as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGR(dim) cells were only partially resistant to apoptosis, whereas hGR(dim4) cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGR(dim4) cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants.     

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort     

Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor

We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.     

Mutations in squirrel monkey glucocorticoid receptor impair nuclear translocation

To identify the determinants of impaired glucocorticoid receptor (GR) signaling in a model of glucocorticoid resistance, cloned GR from Guyanese squirrel monkeys (gsmGR) was tagged with enhanced green fluorescent protein, and nuclear translocation was examined in transfected COS1 cells. In keeping with evidence that gsmGR transactivational competence is impaired, we found that nuclear translocation is likewise diminished in gsmGR relative to human GR (hGR). Experiments with GR chimeras revealed that replacement of the gsmGR ligand binding domain (LBD) with that from hGR increased translocation. Truncated gsmGR constructs lacking the LDB after amino acid 552 also showed increased translocation even in the absence of cortisol. Three back-mutations of gsmGR to hGR (Thr551Ser, Ala616Ser, and Ser618Ala) in the LBD confirmed that these amino acids play a role in diminished translocation.     

A new, lineage specific, autoup-regulation mechanism for human glucocorticoid receptor gene expression in 697 pre-B-acute lymphoblastic leukemia cells

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.     

Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system.

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.     

Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7

The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.     

Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells. Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer.

Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.     

Glucocorticoid receptor represses the Dex-mediated induction of human androgen response element-linked Luc activity

A human androgen response element (hARE), identified within intron 8 of the human sterol regulatory element-binding protein cleavage-activating protein, interacts with both glucocorticoid receptor (GR) and androgen receptors (AR). The aim of this study was to test the hypothesis that human GR (hGR) might modulate the expression of a hARE-linked reporter gene by dexamethasone (Dex). The hypothesis was tested by: a) co-transfecting HepG2 cells with a hGR and a luciferase (Luc)-reporter gene for performing in vitro investigations and b) by their co-injection into the tail vein of mice for in vivo investigation. In vitro co-transfected cells and the in vivo co-injected mice were then treated with Dex. Our results have led us to concluded that both transfection and injection of the hGR leads to a repression in the Dex-mediated induction of hARE-linked Luc activity both in vitro and in vivo settings. These findings suggest that this assay system allows screening of drug candidates affecting to a signal transduction pathway of the GR and AR and may help in the future discovery and analysis of novel and selection of GR and AR agonists.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.