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S S Simons

Publications and source records attributed to S S Simons.

At least 37 records · Page 2Linked to original sources
PubMed1996

Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains.

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins.

Amino Acid Sequence↗
PubMed1996

A negative tyrosine aminotransferase gene element that blocks glucocorticoid modulatory element-regulated modulation of glucocorticoid-induced gene expression.

Tyrosine aminotransferase (TAT) is the prototypic steroid-inducible gene. Recently, we have found that the modulation of TAT induction properties is reproduced by a novel cis-acting TAT gene element, the glucocorticoid modulatory element (GME). This GME lies about 1 kb upstream of the glucocorticoid response elements (GREs) of the TAT gene and binds a heterooligomer of two recently defined proteins. We now report the existence of an additional TAT gene element between the GME and the GREs that blocks the action of the GME and thus prevents the left shift in the glucocorticoid dose-response curve caused by the GME. This negative element has the properties of a silencer because its activity is relatively position- and orientation-independent. The interaction appears to be stoichiometric in that the effects of a single negative element can be overcome by a second GME. This negative element also has an intrinsic inhibitory activity in the absence of the GME. The majority of the negative element activity could be elicited by a 56-bp sequence between -3105 and -3050 bp of the TAT gene. Multiple, clustered mutations of this sequence reduced, but did not eliminate, the negative activity. Further efforts to restrict the negative element were unsuccessful, suggesting that multiple sequences are required for full activity. High affinity, sequence-specific binding of a trans-acting factor(s) was observed in gel shift assays. This binding was half-maximally competed by a 4.4-fold excess of nonradioactive probe and was very stable once formed (delta H [symbol: see text] dissoc. = 32 kcal/mol), suggesting that low concentrations of a high affinity binding protein(s) exist in nuclear extracts. Further support for this conclusion came from the observation that cotransfection of a plasmid containing multiple copies of the 56-bp negative element was able to relieve the negation of GME activity in a GME-56-bp-GRE reporter construct. These data directly support the role of a trans-acting factor(s) in binding to the 56-bp negative element and blocking GME activity. Collectively, these data suggest that glucocorticoid induction of TAT gene expression is subject to multiple levels of control by several new cis-acting elements and thus is much more complex than previously appreciated.

Base Sequence↗
PubMed1995

The factor binding to the glucocorticoid modulatory element of the tyrosine aminotransferase gene is a novel and ubiquitous heteromeric complex.

Glucocorticoid induction of the tyrosine aminotransferase gene deviates from that of many glucocorticoid-responsive genes by having a lower EC50 and displaying more agonist activity with a given antiglucocorticoid. A cis-acting element, located 3646 base pairs upstream of the start of tyrosine aminotransferase gene transcription, has been found to be sufficient to reproduce these variations with heterologous genes and promoters (Oshima, H., and Simons, S.S., Jr. (1992) Mol. Endocrinol. 6, 416-428). This element has been called a glucocorticoid modulatory element, or GME. Others have called this sequence a cyclic AMP-responsive element (CRE) due to the binding of the cyclic AMP response element binding protein (CREB). We now report the partial purification and characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa that bind to the GME/CRE as a heteromeric complex. This purification was followed by the formation of a previously characterized, biologically relevant band in gel shift assays. By several biochemical criteria, the GMEBs differed from many of the previously described CREB/CREM/ATF family members. Partial peptide sequencing revealed that the sequences of these two proteins have not yet been described. Size exclusion chromatography and molecular weight measurements of the gel-shifted band demonstrated that the GMEBs bound to the GME as a macromolecular complex of about 550 kDa that could be dissociated by deoxycholate. Similar experiments showed that CREB bound to the GME as heteromeric complexes of about 310 and 360 kDa. As determined from gel shift assays, GMEB1 and -2 are not restricted to rat liver cells but appear to be ubiquitous. Thus, these novel GMEBs may participate in a similar modulation of other glucocorticoid-inducible genes in a variety of cells.

Amino Acid Sequence↗
PubMed1994

Metal oxyanion stabilization of the rat glucocorticoid receptor is independent of thiols.

The ability of sodium molybdate, both to stabilize the steroid binding activity of glucocorticoid receptors and to prevent the activation of receptor-steroid complexes to a DNA binding species, has long been thought to involve thiols. Two receptor thiols in particular, Cys-656 and Cys-661 of rat receptors, have been suspected. The requirements for the action of molybdate, as well as two other metal oxyanions (tungstate and vanadate) known to exert the same effects as molybdate, have now been examined with receptors in which these thiols, or a third cysteine in the steroid binding cavity (Cys-640), have been mutated to serine. No mutation prevented any metal oxyanion from either stabilizing steroid-free receptors or blocking the activation of complexes for binding to nonspecific or specific DNA sequences. Thus, Cys-640, Cys-656, and Cys-661 are not required for any of the effects of molybdate, tungstate, or vanadate with rat glucocorticoid receptors. Studies with hybrid receptors, and with a 16-kDa steroid binding core fragment containing only 3 cysteines at positions 640, 656, and 661, indicated that no cysteine of the rat receptor was needed to maintain responsiveness to molybdate. Even when all of the thiol groups in crude cytosol were blocked by reaction with excess methyl methanethiol-sulfonate, each metal oxyanion was still able to stabilize the steroid binding of receptors. These results argue that molybdate, tungstate, and vanadate each interact with the receptor or an associated nonreceptor protein(s) in a manner that does not require thiols. An indirect mechanism of molybdate action was evaluated in light of the recent report that the whole cell actions are mediated by increased levels of intracellular cGMP. Under cell-free conditions, however, the effects of molybdate could not be reproduced by cGMP derivatives. Evidence consistent with a direct effect was that molybdate, tungstate, or vanadate each modified the kinetics of proteolysis of wild type receptors at 0 degrees C by trypsin, presumably due to induced conformational changes of the receptor. This alteration of trypsin digestion constitutes yet another effect of metal oxyanions on the glucocorticoid receptor.

Affinity Labels↗
PubMed1994

Absence of intramolecular disulfides in the structure and function of native rat glucocorticoid receptors.

The presence of intramolecular disulfides in different functional states of the native glucocorticoid receptor in the absence of added oxidants has been examined on nonreducing SDS-polyacrylamide gels. Possible disulfides were trapped by the reaction of thiols in crude receptor solutions with methyl methanethiolsulfonate or iodoacetamide. The presence of diffuse bands at lower molecular weights than either the fully reduced or the thiol-blocked species for both the intact 98-kDa receptor and the 42-kDa chymotryptic fragment was diagnostic of an intramolecular disulfide(s) that had undergone thiol-disulfide rearrangements. However, both the rearrangements and the formation of intramolecular disulfides were found to occur only with denatured receptors during gel analysis. It appears that the thiols normally complexed with zinc in the zinc fingers may be recruited for disulfide bond formation. Finally, even when a documented intramolecular disulfide was formed in solutions of the native protein, thiol-disulfide rearrangements did not occur. The tertiary structure of the receptor is thus constituted in a manner that not only limits the formation of disulfides but also prevents the usually facile rearrangements of disulfide bond-containing structures to receptor forms that may have greatly reduced activity. Therefore, although intramolecular disulfide bonds may be of transitory importance, the structural or functional changes of native glucocorticoid receptors that are associated with steroid binding, activation, and dissociation of heat shock protein 90 neither involve nor require the formation or reduction of stable intramolecular disulfides.

Animals↗
PubMed1994

Microtubules are not required for glucocorticoid receptor mediated gene induction.

Steroid-free glucocorticoid receptors are generally considered to reside in the cytoplasm of cells. After the binding of steroids, the receptors translocate into the nucleus in a manner that has been proposed to involve microtubules. However, some results with inhibitors of microtubule assembly argue to the contrary. In all of these studies, only the whole cell localization of receptors has been examined; the biological activity of these receptors has not been determined. We now report that steroid-induced gene expression is maintained in the absence of intact microtubules. This argues that microtubules are not required for either the nuclear translocation or biological activity of glucocorticoid receptors.

Animals↗
PubMed1994

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes.

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700-3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.

Animals↗
PubMed1993

Sequence-selective interactions of transcription factor elements with tandem glucocorticoid-responsive elements at physiological steroid concentrations.

Synergism in transcription is said to occur when the combined response from two DNA elements for the binding of trans-acting factors is greater than the sum of the responses from each element in isolation. The synergism of steroid receptors with themselves or with other trans-acting factors at saturating concentrations of steroid has proved to be an important component of steroid-regulated gene transcription. We have recently described a glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene that, in conjunction with a trans-acting factor, modulates the transcriptional activity of receptor-glucocorticoid and -antiglucocorticoid complexes with homologous and heterologous genes and promoters (Oshima, H., and Simons, S. S., Jr. (1992) Mol. Endocrinol. 6, 416-428). We now report that, under certain circumstances, the GME displays synergistic activity with a glucocorticoid-responsive element (GRE). However, several properties of GME action are different from those previously observed for synergism. The effects of the GME were marked at subsaturating or physiological concentrations of glucocorticoids but insignificant at saturating concentrations, which are the established conditions for synergism. The GME was found to increase the agonist activity of partial antiglucocorticoids, while synergism involving antisteroids has yet to be reported. Furthermore, the GRE was active in conjunction with two tandem repeats of a GRE, which was a combination that did not support conventional synergism. Most importantly, the effects of the GME were greater than with any other trans-acting factor binding element tested, indicative of a sequence-selective activity. The efficacy of the GME was also insensitive to the spacing between elements. Thus, the GME provides a mechanism for selective transcriptional modulation by physiological concentrations of steroid, and by antisteroids, of a common class of genes that are under the control of two or more GREs.

Animals↗
PubMed1993

Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90.

Previously, it has been shown that the hormone binding domain of the glucocorticoid receptor acts as a transferable regulatory cassette that can confer hormonal control onto chimeric proteins [Picard, D., Salser, S. J., & Yamamoto, K. R. (1988) Cell 54, 1073-1080]. The hormone binding domain of the glucocorticoid receptor contains its site of interaction with the 90-kDa heat-shock protein, hsp90 [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. We have now transfected COS cells with cDNAs for fusion proteins containing beta-galactosidase and portions of the glucocorticoid receptor, and we demonstrate a correlation between hormone regulation of fusion protein localization and binding of the fusion proteins to hsp90. The hormone binding domain (residues 540-795) of the rat glucocorticoid receptor is sufficient for conferring hormone regulation onto a fusion protein and for intracellular binding of a fusion protein to hsp90. The hormone binding domain of the rat glucocorticoid or the human estrogen receptor is also sufficient to permit reticulocyte lysate-mediated refolding of a fusion protein into association with hsp90. Consistent with the results of fusion protein localization in intact cells, binding of a fusion protein to hsp90 blocks binding of antibody directed against the NL1 nuclear localization signal of the glucocorticoid receptor. These observations argue strongly that the hormone binding domain of the glucocorticoid receptor confers hormonal control of fusion proteins by conferring hormone-regulated binding to hsp90.

Animals↗
PubMed1993

Differential effects of the reversible thiol-reactive agents arsenite and methyl methanethiosulfonate on steroid binding by the glucocorticoid receptor.

The hormone binding domain of the glucocorticoid receptor contains a unique vicinally spaced dithiol, and when it is bound by arsenite under conditions that are specific for reaction with vicinally spaced dithiols versus monothiols, steroid binding activity is eliminated [Simons, S. S., Jr., Chakraborti, P. K., & Cavanaugh, A. H. (1990) J. Biol. Chem. 265, 1938-1945]. The vicinally spaced dithiol lies in a region of the receptor that appears to be a contact site for hsp90, which is required for the high-affinity steroid binding conformation of the glucocorticoid receptor [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. As part of a long-term project to develop a vicinal dithiol-specific agent that will permit studies of ligand-induced conformational changes in this region of the receptor, we have examined here the differential effects of two reversible thiol-reactive agents, arsenite and MMTS. At low concentration, arsenite inactivates the steroid binding activity of the unliganded receptor in a vicinal dithiol-specific manner, whereas dissociation of steroid from untransformed, transformed, or DNA-bound transformed receptors occurs only at concentrations typical of monothiol interactions. MMTS produces a unique bimodal effect on the steroid binding capacity of the unliganded receptor at pH 9 that is pH-dependent and becomes essentially unimodal at physiological pH. Whereas arsenite disrupts the dexamethasone-receptor complex more readily than the triamcinolone acetonide-receptor complex, MMTS has the opposite effect. During treatment for 1 h at 0 degree C, neither reagent causes dissociation of hsp90 from the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Glucocorticoid receptor binding to rat liver nuclei occurs without nuclear transport.

The binding of cell-free activated glucocorticoid receptor-steroid complexes from HTC cells to various preparations of HTC and rat liver nuclei has been examined under conditions that did or did not support the nuclear translocation of macromolecules via nuclear pores. To the best of our knowledge, this is the first such study with functionally active isolated nuclei. Conventionally prepared HTC nuclei were found to be porous, as determined from their inability to exclude the fluorescent macromolecule phycoerythrin (PE) at 4 degrees C. Thus the nuclear binding of activated complexes to these nuclei can not involve nuclear translocation. Further studies, using established conditions with sealed nuclei prepared from rat liver, revealed that nuclear translocation of PE containing a covalently linked, authentic nuclear translocation sequence could be obtained at 22 degrees C, but not at 4 degrees C. However, under the same conditions, activated glucocorticoid complexes displayed equal levels of nuclear binding at both temperatures. We therefore conclude that the current translocation conditions with intact rat liver nuclei are not sufficient to reproduce the nuclear transport of glucocorticoid complexes observed in intact cells. The nuclear binding that was seen with intact rat liver nuclei was not affected by aurintricarboxylic acid, which selectively inhibits protein-nucleic acid interactions. The antibody AP-64, shown to be specific for amino acids 506-514 of the nuclear translocation sequence of the rat glucocorticoid receptor, inhibited the nuclear binding of activated complexes, apparently by blocking receptor access to the nuclear membrane. Collectively, these data argue that activated complex binding to nuclei capable of nuclear translocation involves only an association with nuclear membrane components such as nuclear pores. Thus this system, and these reagents, may be useful in future studies of activated complex binding to nuclear pores.

Amino Acid Sequence↗
PubMed1993

A new cis-acting element involved in tissue-selective glucocorticoid inducibility of tyrosine aminotransferase gene expression.

Tyrosine aminotransferase (TAT), a prototypical steroid hormone-inducible gene, has been used extensively in studies of tissue-specific control of gene transcription. Over the last several years, a total of five cis-acting elements have been implicated in the tissue-specific expression and induction of the TAT gene in rat liver. These elements are all located upstream of the start of transcription, at -11, -5.5, -3.6, -2.5, and approximately -0.1 kilobases (kb). We now have used both stable and transient transfection assays to define a new element between -2.56 and -2.3 kb that regulates the fold induction by glucocorticoids in a tissue-selective manner. Compared to simple glucocorticoid-regulated constructs, which were used as controls, the major effect of this element was repression of glucocorticoid inducibility in nonliver cells. This activity was both orientation and position independent and was seen with homologous and heterologous promoters and genes. Although this element, therefore, possessed silencer-like activity, it was unable to extinguish gene expression in nonliver cells. In fact, the observance of some glucocorticoid-induced gene expression was additional evidence that the repression derived from an element that is distinct from the glucocorticoid-responsive element at -2.5 kb. A second element was found between -2.95 and -2.56 kb that acts in a tissue nonspecific manner to reduce the absolute level of gene expression in both hepatic and nonhepatic cells. The combined effects of this tissue-nonselective element and the above-mentioned tissue-selective element were to almost completely eliminate glucocorticoid inducibility in nonhepatic cells.

Animals↗
PubMed1992

Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid receptors.

The involvement of a vicinally spaced dithiol group in steroid binding to the glucocorticoid receptor has been deduced from experiments with the thiol-specific reagent methyl methanethiolsulfonate and the vicinal dithiol-specific reagent sodium arsenite. The vicinally spaced dithiol appears to reside in the 16-kDa trypsin fragment of the receptor, which is thought to contain 3 cysteines (Cys-640, -656, and -661 of the rat receptor) and binds hormone with an approximately 23-fold lower affinity than does the intact 98-kDa receptor. We now report that the steroid binding specificity of preparations of this 16-kDa fragment and the intact receptor are virtually identical. This finding supports our designation of the 16-kDa fragment as a steroid-binding core domain and validates our continued use of this tryptic fragment in studies of steroid binding. To identify the cysteines which comprise the vicinally spaced dithiol group, and to examine further the role of cysteines in steroid binding, a total of five point mutant receptors were prepared: cysteine-to-serine for each suspected cysteine, cysteine-to-glycine for Cys-656, and the C656,661S double mutant. Unexpectedly, each receptor with a single point mutation still bound steroid. Even the double mutant (C656,661S) bound steroid with wild type affinity. These results suggest that none of these cysteines are directly required either for steroid binding to the glucocorticoid receptor or for heat shock protein 90 association with the receptor. However, the presence of Cys-656 was obligatory for covalent labeling of the receptor by [3H]dexamethasone 21-mesylate. Studies with preparations of the 98 and 16 kDa forms of these mutant receptors revealed both that Cys-656 and -661 comprise the vicinally spaced dithiols reacting with arsenite and that any two of the three thiols could form an intramolecular disulfide after treatment with low concentrations of methyl methanethiolsulfonate. These data, in conjunction with those from experiments on the effects of steric bulk on various receptor functions, support a model for the ligand binding cavity of the receptor that involves all three thiols in a flexible cleft but where thiol-steroid interactions are not essential for binding.

Affinity Labels↗
PubMed1992

Modulation of the agonist activity of antisteroids by a novel cis-acting element.

The amount of agonist activity displayed by the antiglucocorticoid dexamethasone mesylate (Dex-Mes) for the induction of tyrosine aminotransferase (TAT) in rat hepatoma cells is greater than for glutamine synthetase and varies over a period of weeks. This variation, which has been reproduced over a period of 40 h by changing the density of the cells, suggests the involvement of a trans-acting factor. The target of this proposed trans-acting factor has now been localized to the region between -3.9 to -2.9 of the rat TAT gene from experiments with cells that were stably transfected with hybrid TAT/CAT constructs. Deletion experiments with transiently transfected TAT/tk promoter/CAT constructs revealed that this entire activity could be conveyed by a 21 bp sequence of the TAT gene. Gel shift experiments support the binding of a factor(s) to this 21 bp sequence. Thus the activity of the antagonist Dex-Mes is relatively independent of steroid structure and is largely determined by the further interactions of a trans-acting factor with the cis-acting sequence. We call this novel sequence a glucocorticoid modulatory element. A model is advanced which accounts for almost all of the results concerning TAT induction by glucocorticoids. This same model may also be useful in explaining why the amount of agonist activity of most antisteroids varies, even for different genes within the same cell.

Animals↗
PubMed1992

Modulation of glucocorticoid induction of tyrosine aminotransferase gene expression by variations in cell density.

Glucocorticoids induce tyrosine aminotransferase (TAT) in hepatoma cells. We have previously shown that both the concentration of the agonist dexamethasone (Dex) required for half-maximal induction (EC50) and the amount of agonist activity produced by the antagonist dexamethasone 21-mesylate (Dex-Mes), expressed as a percentage of maximum induction achieved by Dex, are different in Fu5-5 and HTC cells. Furthermore, both activities vary over several weeks in each cell line in an apparently random manner, but, nevertheless, are correlated by a linear semilog plot. We now find that this long term and previously unpredictable variation in both the Dex EC50 and the amount of Dex-Mes agonist activity for the induction of TAT enzyme activity can be made to occur reproducibly in 40 h or less by changing the cell density and/or amount of medium in the tissue culture plates. Thus, a higher cell density and/or a lower volume of medium produced both higher amounts of Dex-Mes agonist activity and lower EC50 values for Dex. Experiments with cells at different densities but exposed to the same medium indicated that cell density was the dominant determinant. A qualitatively identical modulation was seen at the level of TAT mRNA, but not mouse mammary tumor virus RNA. We are not aware of any previous report of cell growth conditions altering either the level of agonist activity of an antisteroid or the EC50 of a full agonist. These results further indicate that extrachromosomal parameters, such as cell-cell contact and/or a diffusable factor(s), can modulate the basic features of glucocorticoid induction of some, but not all, glucocorticoid-inducible genes.

Animals↗