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.     

Interaction of the hepatic glucocorticoid-receptor complex with Affigel blue

Summary Unlike the unactivated glucocorticoid-receptor complex, the thermally activated glucocorticoid-receptor complex was able to bind to Affigel blue (a matrix previously shown to bind proteins containing a dinucleotide fold region) under low ionic conditions (0.05 M KCl). Glucocorticoid-receptor complex binding capacity to Affigel blue was enhanced by increasing salt concentration. Optimal binding was obtained at 0.15 M KCl and remained at a plateau level up to 0.4 M KCl. In contrast to Affigel blue binding, glucocorticoid-receptor complex binding to nuclei was optimum at low ionic strength buffer, declined at 0.15 M KCl and became negligible at 0.4 M KCl. Interestingly, at physiological ionic strength (0.15 M KCl) both nuclei and Affigel blue bound to the glucocorticoid-receptor complex with almost identical capacity. Glucocorticoid-receptor complexes incubated 45 min at 25 °C (activation conditions) in the presence of 10 mM molybdate were unable to bind to Affigel blue (or isolated nuclei) as expected. The results obtained suggest that Affigel blue mimics isolated nuclei in the binding of activated glucocorticoid-receptor complexes under physiological (0.15 M KCl) conditions. In addition, Affigel blue may provide a rapid and easy method to study glucocorticoid-receptor complex activation and interaction with nuclear acceptor sites.     

Purification, characterization, and activation of the glucocorticoid-receptor complex from rat kidney cortex

The unactivated molybdate-stabilized glucocorticoid receptor (GcR) was purified from rat kidney cortex cytosol (RKcC) by using a modification of the procedure previously described by this laboratory for rat hepatic receptor. The purification includes affinity chromatography, gel filtration, and ion-exchange chromatography. The final preparation (approximately 1000-fold pure as determined from specific radioactivity) was used in subsequent physicochemical and functional analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single heavily Coomassie-stained band at 90 kilodaltons. Density gradient ultracentrifugation indicated a sedimentation coefficient of 10.5 +/- 0.05 S (n = 2). Chromatography on an analytical gel filtration column produced a Stokes radius (Rs) of 6.4 +/- 0.07 nm (n = 5). The Rs was unchanged when the molybdate-stabilized GcR was analyzed in the presence of 400 mM KCl or when analyzed in the unpurified (cytosolic) state. In contrast, the hepatic GcR was observed to exist as a larger form in cytosol (7.7 +/- 0.2 nm). Following purification, or upon gel filtration analysis under hypertonic conditions, the Rs was similar to that of the unpurified RKcC GcR. Following removal of molybdate from RKcC GcR and thermal activation (25 degrees C/30 min), DNA-cellulose binding increased 1.5-2-fold over the unheated control. Addition of RKcC or hepatic cytosol (endogenous receptors thermally denatured at 90 degrees C/30 min or presaturated with 10(-7) M radioinert ligand) during thermal activation increased DNA-cellulose binding an additional 2-6-fold beyond the heated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).     

Interaction of sodium thiocyanate with rat hepatic glucocorticoid-receptor complexes

0.1–0.3 M sodium thiocyanate greatly enhanced the rate of inactivation of unbound rat hepatic glucocorticoid receptors in vitro at 4°C. Prior treatment of the unbound glucocorticoid receptor with 10 nM molybdate (at 25°C for 30 min) protected the receptor from 0.3 M KCl, but not from 0.3 M NaSCN inactivation. When the [³H]dexamethasone-receptor complex was examined on sucrose density gradients containing 0.1 M NaSCN, the receptor sedimented as a 4 S complex rather than the 7 S form observed in 0.1 M KCl gradients. NaSCN was found to be more effective in the extraction of both in vivo and in vitro nuclear-bound [³H]dexamethasone-receptor complexes than KCl. At a concentration of 0.3 M, NaSCN extracted most of the specific nuclear-bound receptor. 50 mM NaSCN significantly blocked the thermal activation of preformed [³H]dexamethasone-receptor complexes. The chaotropic salt, NaSCN, appears therefore to have significant effects on glucocorticoid receptors in vitro. In addition, NaSCN appears to be a useful agent in quantitative extraction of steroid from nuclear-bound steroid-receptor complexes.     

Hydrodynamic and biochemical correlates of the activation of the glucocorticoid-receptor complex

Possible changes in the size and shape of the glucocorticoid-receptor complex (GRC) following activation remain poorly documented, due to the lability and possible activation of the receptor during the determination of these hydrodynamic parameters. In the present study molybdate was used to stabilize the GRC, thus preventing these uncontrolled transformations. Cytosol prepared from mouse whole brains was incubated for 18 h at 0-2 degrees C with [3H]triamcinolone acetonide (+/- molybdate). Activation was then initiated by incubation at 22 degrees C for variable times and quenched at 0 degree C by adding molybdate. The Stokes radius and sedimentation coefficient of the GRC declined from 77 A and 9.2 S before activation to 58 A and 3.8 S after activation. These measurements remained consistent after recycling GRC between sedimentation and gel filtration procedures and correspond to a 3-fold reduction in the relative molecular mass. The loss and formation of the 297 and 92 kDa species, respectively, after different durations of activation correlated nearly perfectly with increased binding of GRC to DNA-cellulose (DNA-C). The observed size change also correlated well with decreased adsorption to DEAE-cellulose filters (DE-81) and increased adsorption to glass fiber filters (GF/C). The increased adsorption to GF/C may reflect an increase in hydrophobicity which, with extended durations of activation, leads to increased aggregation and reduced binding to DNA-C, but not to a change in adsorption to DE-81. We propose that during activation the 297 kDa form of the GRC splits to form a 92 kDa species that displays an increased affinity for DNA.     

Activation of the glucocorticoid-receptor complex

A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid-receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid-receptor complexes to bind to polyanions, such as DNA-cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)-Sephadex or DEAE-cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid-receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5'-phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid-receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA-cellulose. The ability of 1,10-phenanthroline, a metal chelator, to inhibit the DNA-cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance.     

Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation

We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.     

Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated.

Molybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with 1 M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique. Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.     

The "activated" hepatic glucocorticoid-receptor complex. Its generation and properties.

The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex.     

The activated hepatic glucocorticoid-receptor complex: A simple one-step method for its separation from nonactivated complex

Protamine sulfate was found to precipitate completely the nonactivated [³H]-dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [³H]dexamethasone-receptor complex, incubated at 0–4°C for 2 hr, resulted in the complete precipitation of [³H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 mm CaCl₂ to the cytosol resulted in the appearance of activated [³H]dexamethasone-receptor complex in the supernatant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by pI. Protamine sulfate could not affect the separation of activated [³H]dexamethasone-receptor complex at salt concentrations above 100 mm NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [³H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoid-receptor activation.     

Aqueous two-phase partition studies of the glucocorticoid receptor activation and pH-dependent changes of rat liver glucocorticoid-receptor complex in partly purified preparations

• 1.1. The activation of the glucocorticoid receptor of rat liver cytosol was studied by the use of partitioning in an aqueous dextran-poly(ethylene glycol) two-phase system.
  • 1.1.a, Incubation under conditions generally found to stimulate the activation results in a time-dependent increase of the partition coefficient of the glucocorticoid-receptor complex in the two-phase system.
  • 1.2.b, This increase was found to result from the fact that the activated form of the complex has a higher partition coefficient that the nonactivated one, since the two forms are eluted from DNA-Sepharose columns as clearly separated peaks with different partition coefficients, and a redistribution of complex between the peak occurs without any changes of the partition coefficient of each single peak.
  • 1.3.c, The partition coefficient of a partly purified nonactivated complex was found to follow the same time course towards a higher value under ‘activating’ incubation conditions as the complex in whole cytosol.
• 2.2. The dependence of the partition coefficient of the glucocorticoid-receptor complex on the pH of the two-phase system revealed a pH-dependence of the state of the complex. Experiments with partly purified preparations of the complex showed that this change was either a conformational change or an aggregation of high affinity of the complex with itself or another DNA-binding substance.

     

Effects of sodium tungstate on the nuclear uptake of glucocorticoid-receptor complex from rat liver

Effects of sodium tungstate on the nuclear uptake of rat liver cytosolic glucocorticoidreceptor complex were examined at pH 7. The nuclear uptake of heat-activated [³H]triamcinolone acetonide-receptor complex was blocked completely in the presence of 1 mm tungstate. A preincubation of nuclear preparation with tungstate (>0.1 mm) blocked the subsequent uptake of [³H]triamcinolone acetonide-receptor complex. When the tungstate-treated nuclear preparation was washed with 0.3 M KCl, its [³H]triamcinolone acetonide-receptor complex binding capacity recovered to 50% of that of control samples with no tungstate treatment. A preincubation of chromatin with tungstate yielded similar results. The nuclear-bound [³H]triamcinolone acetonide-receptor complex, formed either by an in vivo administration of [³H]triamcinolone acetonide or by an in vitro incubation of glucocorticoid-receptor complex with isolated nuclei, was extracted by tungstate in a concentration-dependent manner. The majority of nuclear-bound [³H]triamcinolone acetonide could be extracted with 0.1 and 1 mm tungstate from in vitro- and in vivo-labeled nuclei, respectively. The tungstate-extracted steroid-receptor complexes sedimented in 4–5 S and 3.3–3.5 S region in 10 mm KCl- and 0.3 mm KCl-containing sucrose gradients, respectively. Tungstate treatment caused an irreversible loss of the nuclear binding capacity of [³H]triamcinolone acetonide-receptor complex which could not be recovered after dialysis. These studies indicate that tungstate affects both glucocorticoidreceptor complex and certain nuclear or chromatin proteins.     

Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes

Bovine pancreatic ribonuclease (RNase) A and S protein (enzymatically inactive proteolytic fragment of RNase A which contains RNA binding site) stimulate the activation, as evidenced by increasing DNA-cellulose binding, of highly purified rat hepatic glucocorticoid-receptor complexes. These effects are dose dependent with maximal stimulation of DNA-cellulose binding being detected at approximately 500 micrograms (50 units of RNase A/mL). RNase A and S protein do not enhance DNA-cellulose binding via their ability to interact directly with DNA or to increase nonspecific binding of receptors to cellulose. Neither S peptide (enzymatically inactive proteolytic fragment which lacks RNA binding site) nor cytochrome c, a nonspecific basic DNA binding protein, mimics these effects. RNase A and S protein do not stimulate the conformational change which is associated with activation and is reflected in a shift in the elution profile of receptor complexes from DEAE-cellulose. In contrast, these two proteins interact with previously heat-activated receptor complexes to further enhance their DNA-cellulose binding capacity and thus mimic the effects of an endogenous heat-stable cytoplasmic protein(s) which also function(s) during step 2 of in vitro activation [Schmidt, T. J., Miller-Diener, A., Webb, M. L., & Litwack, G. (1985) J. Biol. Chem. 260, 16255-16262]. Preadsorption of RNase A and S protein to an RNase affinity resin containing an inhibitory RNA analogue, or trypsin digestion of the RNA binding site within S protein, eliminates the subsequent ability of these two proteins to stimulate DNA-cellulose binding of the purified receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of rat renal phosphate-dependent glutaminase coupled to Sepharose. Evidence that dimerization is essential for activation.

In the absence of phosphate, purified rat renal phosphate-dependent glutaminase exists as a catalytically inactive protomer. The addition of phosphate results in both dimerization and activation of the glutaminase. Covalent attachment of the dimeric form of the glutaminase to CNBr-activated Sepharose was achieved with 84% retention of activity. At least 70% of the bound glutaminase activity was expressed even in the absence of added phosphate. In addition, 6-diazo-5-oxo-L-norleucine, which interacts only with the catalytically active form of the glutaminase, inactivates the bound dimeric form of glutaminase at the same rate in either the absence or the presence of added phosphate. Therefore retention of dimeric structure is apparently sufficient to maintain glutaminase activity. In contrast, the coupling of the protomeric form of the enzyme to Sepharose resulted in retention of only 3% of the phosphate-induced glutaminase activity. However, up to 48% of this activity could be reconstituted by addition of soluble glutaminase under conditions that promote dimerization. These results indicate that the monomeric form of the glutaminase has minimal inherent activity and that dimerization is an essential step in the phosphate-induced activation of the glutaminase.     

The effect of alkaline phosphatase on the activation of glucocorticoid-receptor complexes in rat liver cytosol

Calf intestinal alkaline phosphatase was found to stimulate the rate of in vitro activation of rat liver glucocorticoid-receptor complexes. This effect was registered both at 0 and 25 degrees C and could be prevented by sodium molybdate. The resulting change in sedimentation behaviour (shift of sedimentation coefficient from 9.6 S to 4.8 S for molybdate-stabilized and alkaline phosphatase-treated complexes, respectively) was similar to that observed after heat activation.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Evidence suggesting that the life span of Mycobacterium avium complex is longer than that of other mycobacteria

Mycobacterium avium complex strains often contain considerably more numbers of viable bacterial units per mg wet weight than other mycobacteria, especially other slowly growing ones. This finding suggests that the life span of M. avium complex strains is often longer than the life span of other mycobacteria. The other mycobacteria, especially slowly growing ones seem to die more rapidly after their multiplication.