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G Litwack

Publications and source records attributed to G Litwack.

At least 73 records · Page 4Linked to original sources
PubMed1992

A novel, highly conserved structural motif is present in all members of the steroid receptor superfamily.

Steroid and thyroid hormone receptor superfamily members are ligand potentiated transcription factors. Recent evidence indicates that one aspect of steroid receptor action is an interaction with other trans-acting factors, such as the glucocorticoid receptor with the AP1 transcription factor, for example. Using a structural approach to identify domains of the glucocorticoid receptor responsible for interactions with affiliated transacting factors and DNA, we have identified a putative helix-turn-zipper motif that is conserved in all steroid, thyroid hormone, retinoic acid, and vitamin-D3 receptors. This structural motif is also conserved among new members of the family, the peroxisome proliferator-activated receptors and the retinoid-X receptors. This structural domain is characterized by a pair of amino acids (I,L,V)P that is conserved in all superfamily members. Additional characteristics include six heptad repeats of hydrophobic amino acids, four of which form a canonical leucine zipper in the rat glucocorticoid receptor. Although this leucine repeat is not absolutely conserved among superfamily members, the periodicity of hydrophobic residues is conserved throughout. Based on sequence analyses from the GenEMBL and SwissProt databases using the Genetics Computer Group and MacVector sequence analysis software packages, and the Brookhaven structural database, we present evidence for a novel structural domain, a helix-turn-zipper that is conserved in all superfamily members, and may function in transactivation of cognate genes.

Amino Acid Sequence↗
PubMed1992

Characterization of in vitro translated human mineralocorticoid receptor. Structure and activation.

The structures of the unactivated and activated mineralocorticoid receptors have been difficult to characterize because of receptor lability and steroid dissociation. Therefore, human mineralocorticoid receptor mRNA was translated in rabbit reticulocyte lysate in the presence and absence of [35S]methionine to compare the structure of [3H]aldosterone-bound and [35S]labeled receptor. In vitro synthesized receptor was able to specifically bind [3H]aldosterone. Unactivated [3H]aldosterone-bound and 60% of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 250 mM salt and had a Rs of 72A. Forty percent of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 54A. SDSPAGE showed intact hMR was present in both DEAE-cellulose eluates as three bands between M(r) 110,000-120,000. However, the low salt eluate contained less intact receptor and more lower MW bands. Neither [3H]aldosterone-bound nor [35S]labeled receptor was activated by incubation at 25 degrees C as readily as glucocorticoid receptor studied under identical conditions. Activated [3H]aldosterone-bound receptor eluted from DEAE-cellulose at 100 mM salt and had a Rs of 37A. After activation, 60% of [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 91A. SDS-PAGE of the high and low salt DEAE-cellulose eluates showed that 50% of intact receptor eluted in the low salt peak after activation. These data indicate that: 1. Some in vitro synthesized mineralocorticoid receptor assembles into the heteromeric unactivated form; 2. The remaining intact receptor remains monomeric and unable to bind steroid; 3. Activation causes dissociation of intact receptor from a larger complex; and 4. Activated receptor tends to aggregate.

Aldosterone↗
PubMed1991

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.

Aldosterone↗
PubMed1991

Endogenous modulators of glucocorticoid receptor function also regulate purified protein kinase C.

Modulator-1 and -2, proposed to be novel ether-linked aminophosphoglycerides, were originally identified as regulators of glucocorticoid receptor function (Bodine, P. V., and Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554). We now demonstrate that these modulators are also potent new stimulators of protein kinase C activity in vitro. These endogenous biomolecules regulate purified protein kinase C activity in a biphasic and dose-dependent pattern, as determined by histone phosphorylation. Modulators, at concentrations within their apparent cellular range, stimulate protein kinase C-catalyzed histone phosphorylation 2-4-fold when added separately, or 10-12-fold when added together. This enhancement of kinase activity apparently is specific for protein kinase C, since neither protein kinase M, nor cAMP-dependent protein kinase A are stimulated by the modulators. The stimulation of purified protein kinase C occurs only when the enzyme has been initially activated by calcium, phosphatidylserine, and diacylglycerol, indicating that the modulators do not simply substitute for one of the enzyme cofactors. In addition, the modulators appear to interact directly with protein kinase C, perhaps with the regulatory domain of the enzyme, since these biomolecules inhibit the binding of phorbol ester to purified protein kinase C. Finally, time-course studies of protein kinase C-catalyzed histone phosphorylation indicate that the velocity of the enzyme reaction is increased by the modulators. Taken together, these results suggest that the modulators are a new class of regulators of protein kinase C.

Animals↗
PubMed1991

Characterization and purification of a functional rat glucocorticoid receptor overexpressed in a baculovirus system.

The structure-function relationship of the oligomeric unactivated glucocorticoid receptor is not fully understood. An essential step in the process of understanding such a relationship involves the production of large quantities of the receptor. Using a baculovirus expression system we have been able to overproduce a recombinant rat glucocorticoid receptor (rGR). A cDNA coding for the entire rGR was introduced into the genome of the wild type baculovirus, Autographa californica nuclear polyhedrosis virus, by an in vivo recombination event. Based on specific steroid binding, insect cells infected with the recombinant baculovirus expressed 1-3 x 10(6) receptor molecules/cell which is 15-45 times more than that expressed normally in a hepatocyte. The recombinant rGR expressed in insect cells is indistinguishable from the bona fide rGR with respect to immunogenic reactivity, cytoplasmic localization, sedimentation, chromatographic and electrophoretic mobility, and hormone and DNA binding. Furthermore, the recombinant rGR is expressed as a functional protein as demonstrated by its ability to specifically bind a glucocorticoid agonist, to translocate from the cytoplasm to the nucleus upon hormone-binding, and to act as a transcriptional enhancer. Pulse labeling of recombinant baculovirus-infected insect cells with 32Pi and isolation of the labeled products by immunoprecipitation demonstrated that the recombinant rGR is a phosphoprotein. Thus, the recombinant rGR expressed in insect cells is biologically active and is suitable for structural and functional analysis. A simple three-step purification procedure of the unactivated recombinant rGR is described.

Animals↗
PubMed1991

Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate.

The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.

Animals↗
PubMed1991

Evaluation of synthetic novel ether aminophosphoglycerides for glucocorticoid-receptor complex modulator activity.

Modulator is an endogenous low-mol wt regulator of the glucocorticoid-receptor complex. Structural analysis of purified modulator suggested that it was a novel ether aminophosphoglyceride (Bodine and Litwack 1988b). Analogs of the putative modulator structure have now been synthesized. The synthetic compounds are 1-O-(6-carboxylhexyl)-glycero-3-phosphoserine and the sn-2-methoxy and sn-1-ethylester derivatives. Like modulator, these novel synthetic compounds are water soluble. However, thin-layer chromatography and spectroscopic analysis of these phosphoglycerides indicated significant structural differences between modulator and the synthetic analogs. In particular, the chromatographic behavior of the compounds suggests that modulator is more highly charged than the synthetic derivatives. The synthetic compounds, as well as lysophosphatidylserine, were also tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. None of the analogs exhibited modulator activity in these assays. However, the synthetic compounds were generally less detrimental to receptor steroid-binding than lysophosphatidylserine. From this work, we conclude that although modulator is not mimicked by one of these synthetic phosphoglycerides, a starting point for future structure-function studies has nonetheless been established.

Amines↗
PubMed1990

Glucocorticoid receptor monoclonal antibodies define the biological action of RU 38486 in intact B16 melanoma cells.

The mechanism of action of the synthetic glucocorticoid antagonist, RU 38486, has yet to be completely elucidated. Although RU 38486 is a potent antiglucocorticoid in vivo, several studies have indicated that it has some agonist activities in vitro, such as high-affinity steroid binding to the receptor, activation, and DNA binding. Nevertheless, these in vitro postbinding events do not lead to any known gene expression. To understand the action of the glucocorticoid antagonist RU 38486, we studied glucocorticoid receptor localization on a mouse melanoma cell line (B16C3) by indirect immunofluorescent staining techniques, using monoclonal antibodies to the glucocorticoid receptor. Our data in intact cells suggest that, unlike glucocorticoid agonists such as triamcinolone acetonide, and similar to the glucocorticoid antagonist cortexolone, RU 38486-bound receptors do not translocate to the nucleus and hence do not allow for transcription of glucocorticoid-regulated genes to occur. Passage through the nuclear membrane may be a rate-limiting step in the action of glucocorticoid antagonists, and translocation may in itself be an important regulatory mechanism of steroid hormone action.

Animals↗
PubMed1990

Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s).

Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.

Amsacrine↗
PubMed1990

Purification and characterization of two novel phosphoglycerides that modulate the glucocorticoid-receptor complex. Evidence for two modulator binding sites in the occupied/unactivated steroid hormone receptor.

Modulator is a novel ether aminophosphoglyceride that is commonly known as the low-molecular weight inhibitor of glucocorticoid-receptor complex activation. An ultra-large scale purification of modulator has been performed from 1000 rat livers. This purification was similar to our previous one (Bodine, P. V., and Litwack, G. (1988) J. Biol. Chem. 263, 3501-3512), but involved the chromatography of heated rat liver cytosol on a 7-liter bed volume Sephadex G-15 gel filtration column. Two peaks of modulator activity eluted from the giant gel-filtration column, and these two modulators (peak-1 and peak-2) were chromatographed separately on Dowex-1 anion-exchange columns. Both modulators were determined to be homogeneous after this step by analytical high-performance thin-layer chromatography, analytical high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. Furthermore, although peak-1 and peak-2 differed in molecular weight, the two modulators co-chromatographed by anion-exchange, high-performance thin-layer, and high-performance liquid chromatography. These results suggest that the two modulators have similar structures and therefore appear to be isoforms of each other. In addition, both of the modulators are organic molecules that are devoid of molybdenum and 62 other metals. Activity assays indicated that the larger peak-1 modulator was five times more potent than the smaller peak-2 modulator at inhibiting receptor activation and at stabilizing the steroid-binding ability of the occupied and unoccupied receptors. Mixing experiments indicated that the activities of the two modulators were synergistic for both receptor activation inhibition and for occupied receptor steroid-binding stabilization. However, the effects of peak-1 and peak-2 modulator on unoccupied receptor steroid-binding stabilization were additive. Thus, although the two modulators have similar chemical structures, the biological potencies of the two compounds are different. Moreover, these results suggest that although the unoccupied/unactivated receptor has only one modulator binding site, the occupied/unactivated receptor has two modulator binding sites, one site for each of the isoforms.

Animals↗
PubMed1989

Glucocorticoid-induced lymphocytolysis is not mediated by an induced endonuclease.

The mechanism of glucocorticoid-induced internucleosomal DNA cleavage and cytolysis of lymphatic cells is not known. Recent data (Compton, M.M., and Cidlowski, J.A. (1987) J. Biol. Chem. 262, 8288-8292) suggested that in vivo treatment of rat thymocytes with glucocorticoids induces a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis. In this paper, the possibility that lymphocytolysis may result from glucocorticoid-induced nuclease(s) was examined. Using the rat thymocytes as a model system, we have shown by electrophoretic, enzymatic, and amino acid sequence analysis that the putative glucocorticoid-induced nucleases identified recently by Compton and Cidlowski are in fact H1, H1(0), and core histones, and their gross appearance is not the result of new histone protein synthesis, but a result of the release of histone-containing nucleosomes during chromatin breakdown. Evidence presented here shows that the putative induced nuclease activity is an artifact of the assay system employed. Because our data do not support induction of a glucocorticoid-induced nuclease(s), we examined the possibility that DNA cleavage might be induced by activation of a constitutive endogenous endonuclease. We have shown that it is possible to produce characteristic internucleosomal DNA cleavage of rat thymocytes, merely by incubating intact nuclei from untreated adrenalectomized rat thymocytes with Ca2+ and Mg2+ for a short period of time. However, in glucocorticoid-sensitive human CEM-C7 lymphocytes activation of internucleosomal DNA cleavage was independent of calcium uptake. We conclude that glucocorticoid induction of internucleosomal DNA fragmentation does not necessarily require expression of a new nuclease(s), but is the result of the activation of a constitutive endogenous endonuclease(s). Also, our data suggest that the mechanism which controls activation of internucleosomal DNA cleavage in rat thymocytes differs from that which operates in CEM-C7 lymphocytes.

Animals↗
PubMed1988

Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride.

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.

Animals↗
PubMed1988

Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Molybdate and modulator inhibit receptor activation as measured by DNA-cellulose binding, DEAE-cellulose chromatography, and Sepharose 4B gel filtration. (ii) The ability of molybdate and modulator to inhibit receptor activation and stabilize the unoccupied receptor appears to be additive. (iii) Scatchard analysis of heat-destabilized unoccupied receptors indicates that the number of steroid-binding sites is reduced during destabilization, whereas the steroid dissociation constant remains unchanged. Molybdate and modulator stabilize the receptor by maintaining the number of steroid-binding sites. (iv) Molybdate and modulator do not inhibit alkaline phosphatase-induced destabilization of the unoccupied receptor. However, alkaline phosphatase-induced destabilization is reversed by the addition of dithiothreitol in the presence, but not in the absence, of molybdate or modulator. These results suggest that the mechanism of action for modulator is identical to that of sodium molybdate, and we propose that modulator is the endogenous molybdate factor for the glucocorticoid receptor.

DNA↗
PubMed1987

Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor.

Monoclonal antibodies to the rat hepatic glucocorticoid receptor (GR) were produced by using 4000-fold-purified unactivated rat hepatic GR as the immunogen in an immunization in vitro. Hybridomas were screened for anti-GR antibody production by using an enzyme-linked immunosorbent assay. The antibody, 3A6, described here, is an IgM (lambda). The interaction of 3A6 with the purified GR was explored by sedimentation analysis, where a shift of the 9 S GR to a form with a higher s20,w value was demonstrated. Binding specificity and sensitivity were demonstrated by protein immunoblotting. 3A6 cross-reacted with all rat tissue glucocorticoid receptors (GRs) examined, except those of the brain. Species cross-reactivity was observed with other mammalian GRs (from human CEM-C7 cells and from pig and mouse liver). Immunocytochemical localization of the GR was assessed by indirect immunofluorescence in intact fixed cells, which demonstrated intense cytoplasmic staining in the absence of pretreatment with glucocorticoids and nuclear localization when cells were pretreated with glucocorticoids. This monoclonal antibody significantly inhibited steroid binding to unoccupied receptor and DNA binding of activated steroid-receptor complexes. Furthermore, preincubation of the purified activated GR complex with 3A6 prevented phosphorylation of the GR in vitro. Thus 3A6 differs from previous monoclonal antibodies to the GR in its capacity to cross-react with the human GR and by its specificity for an epitope on or near a functional domain of the GR.

Animals↗
PubMed1986

Evidence for an association of a ribonucleic acid with the purified, unactivated glucocorticoid receptor.

The unactivated glucocorticoid-receptor complex (GRC) was purified from rat hepatic cytosol (approximately 4000-fold by specific activity) by a procedure developed in our laboratory. Following elution of unactivated GRC from DEAE-cellulose with a 0.05-0.5 M potassium phosphate gradient, a second gradient of 0.5-1.0 M potassium chloride was started. This gradient eluted material at 0.6 M potassium chloride that incorporated [32P] in vivo and stained with ethidium bromide. A predominant ethidium bromide stained band of 100-110 nucleotides was observed. The presence of this material was dependent on the presence of highly purified GRC since presaturation of cytosol with 50 microM unlabeled triamcinolone acetonide (TA) precluded the appearance of this material. Experiments with partially purified GRC from CEM-C7 cells incubated in vivo with [14C]uridine indicated that the material eluted at 0.6 M potassium chloride incorporated [14C]uridine. Collectively, these data suggest that a RNA is associated with the purified, unactivated form of the GRC.

Animals↗