The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Characterization of rat ovarian lutropin receptor. Role of thiol groups in receptor association

Rat ovarian lutropin receptor occurs predominantly as a monomer of an apparent molecular mass of 70 or 80 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing and reducing conditions, respectively. The receptor contains 0.4% free cysteine and 1.9% cysteine as cystine, determined by amino acid analysis of the S-carboxymethyl receptor prepared before and after reduction. The presence of free thiol groups was further shown by the specific adsorption of the receptor on p-chloromercuribenzoate-agarose and its susceptibility to 3H labeling with [3H]N-ethylmaleimide or [3H]iodoacetic acid. The receptor readily undergoes association into homo-oligomers. Evidence suggests that the association was caused by the intermolecular oxidation of the free -SH groups to form disulfide bonds. The aggregation could be induced by H2O2 or molecular O2 and was inhibited by sulfhydryl protecting agents such as N-ethylmaleimide, iodoacetic acid, dithiothreitol, cysteine, and Zn(II). The oligomers could be dissociated by reduction into a monomer. 125I-Labeling of the S-carboxymethyl- or N-ethylmaleyl receptor gave a single band of molecular mass 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Furthermore, S-alkylation of the receptor did not affect its binding to the ligand. On reduction, however, it lost its ability to bind to the ligand, but the reduced receptor retained its ability to bind to a specific polyclonal rabbit antireceptor antibody indicating the separation of the ligand and antibody binding sites. Endoproteinase Glu-C cleaved the receptor at a single glutamyl residue to give two components, 46 and 36 kDa. The 36-kDa component was extracellularly located since it contained the carbohydrate. On deglycosylation with endoglycosidase F, it yielded two components, 27 and 25 kDa. The deglycosylation of the reduced intact receptor (80 kDa) with endoglycosidase F occurred in two steps giving 73- and 64-kDa polypeptides, indicating the presence of about 20% carbohydrate contained in two or more N-linked chains.     

Binding of the glucocorticoid receptor to the rat liver nuclear matrix. The role of disulfide bond formation

The nuclear matrix is a putative skeletal structure which has been implicated in many nuclear functions. To assess a possible role of the nuclear matrix in glucocorticoid action, purified rat liver nuclei containing glucocorticoid-receptor complexes were treated with DNase I +/- RNase A followed by 1.6 M NaCl, thus yielding salt-extractable and salt-resistant (nuclear matrix) fractions. The subnuclear distribution of hormone-receptor complexes was determined by following the fate of unmetabolized radiolabel after injection of labeled triamcinolone acetonide into adrenalectomized animals and subjecting various subfractions to immunoblotting using a monoclonal antibody which recognizes the glucocorticoid receptor. Both techniques indicated that 50-70% of the total nuclear hormone-receptor complexes were recovered in the nuclear matrix fraction. Previous results (Kaufmann, S. H., and Shaper, J. H. (1984) Exp. Cell Res. 155, 477-495) suggest that a variety of nuclear polypeptides become nuclease- and salt-resistant as a result of the formation of intermolecular disulfide bonds. The following evidence suggests that disulfide bonds mediate the association between the glucocorticoid receptor and the nuclear matrix. When nuclei were isolated in the absence of sulfhydryl-blocking and -cross-linking reagents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the receptor was present as a high molecular weight disulfide-cross-linked complex. When nuclei were isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, the disulfide bonds which cross-linked the receptor into high molecular weight complexes were absent; and 85-100% of the hormone-receptor complexes were salt-extractable. When nuclei (isolated in the absence of iodoacetamide) were treated with the sulfhydryl-cross-linking reagent sodium tetrathionate, greater than 95% of the nuclear hormone-receptor complexes became resistant to extraction with nucleases and 1.6 M NaCl. The implications of these results for other matrix-associated nuclear functions are discussed.     

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.     

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Purification of the neurotensin receptor from bovine brain

The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of [3,11-tyrosyl-3,5-3H]neurotensin with an apparent affinity (Kd = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of Mr 72,000. Under nonreducing conditions the apparent Mr, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogeneous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.     

Influence of liposome charge and composition on their interaction with human blood serum proteins

The roles of sulfhydryl and disulfide groups in the specific binding of synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membrane preparations from the rat cerebral cortex have been examined. Various sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and methyl methanethiosulfonate (MMTS) inhibited the specific binding of [³H]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. About 80–95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding affinity (K_d) and capacity (B_max). On the other hand, DL-dithiothreitol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [³H]CP-55,940 to the receptor and about 50% inhibition was obtained at a 5 mM concentration. Furthermore, 5mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [³H]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essential to the binding of the ligand to the receptor. In addition, the inhibition of the binding by DTT implies that besides free sulfhydryl group(s), the integrity of a disulfide bridge is also important for [³H]CP-55,940 binding to the cannabinoid receptor.     

Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity

DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation than was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [3H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [3H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. We concluded that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to a nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since we were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), we speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state.     

Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli

We purified and characterized a soluble human interferon gamma receptor expressed in Escherichia coli. The soluble receptor comprises the amino acids 15-246 of the encoded protein (Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280) and was purified from large scale fermentations through four chromatographic steps with an overall recovery of 28%. The refolded soluble receptor shows some heterogeneity on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where it appears as the major band of 27 kDa molecular mass, accompanied by a few minor bands with molecular masses between 26 and 30 kDa. On reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as a homogeneous protein of 32 kDa molecular mass. The soluble interferon gamma receptor is an active and stable protein and is recognized by specific antibodies raised against the native receptor. When nonreduced it has the capacity to specifically bind interferon gamma and to compete for the binding of interferon gamma to the cell surface receptor. The observed heterogeneity of the soluble interferon gamma receptor under nonreducing electrophoretic conditions is probably due to different conformational forms resulting from the formation of non-native intramolecular disulfide bonds among the 8 cysteine residues present in the soluble interferon gamma receptor molecule.     

A microsomal endopeptidase from liver that preferentially degrades stearoyl-CoA desaturase

A protease was purified some 700-fold from rat liver microsomes by a combination of differential detergent solubilization, hydroxyapatite column chromatography, and gel filtration. The protease exhibits substrate selectivity for stearoyl-CoA desaturase (SCD). The purified protease rapidly degraded SCD while other microsomal proteins including cytochrome b(5) and 11beta-hydroxysteroid dehydrogenase were degraded slowly or not at all. The isolated form of the protease has an apparent molecular mass of approximately 90 kDa. Upon incubation, the 90 kDa form of the protease undergoes rapid conversion to a series of smaller proteins. This conversion is associated with a marked increase in proteolytic activity. Diisopropyl phosphofluoridate (DFP) at high concentration partially inhibited the protease activity. The [(3)H]DFP-labeled protease was detected as three protein bands of approximately 66 kDa under nonreducing conditions and a single 25 kDa band under reducing conditions. The purified protease was inhibited by dithiothreitol, suggesting the presence of an essential disulfide bond. These results further define the mechanism by which SCD is rapidly and selectively degraded in isolated liver microsomes.     

Disulfide bonds within the alpha-subunit of insulin receptors in rat liver and brain membranes

We have characterized inter- and intrasubunit disulfide bonds of insulin receptors using reductant-treated rat liver and brain membranes. In autoradiograms of 125I-insulin cross-linked to both membranes pretreated with dithiothreitol, the intensity of affinity-labeled bands of the alpha beta-heterodimer and alpha-subunit was increased. Interestingly, labeled 120 and 110 kDa bands considered to be the alpha-subunit in partially reduced liver and brain membranes moved to 130 and 120 kDa bands under further reduced conditions, respectively. Double electrophoresis of each partially reduced band in the presence of reductants clearly demonstrates that the alpha-subunit of insulin receptors contains intrasubunit disulfide bonds.     

Evidence for Disulfide Bonds in Membrane-Bound and Solubilized Opioid Receptors

The effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane-bound and digitonin-solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D-Ala2-D-Leu5-enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin-solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2. [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.     

Ligand-induced variations in the reactivity of thio groups of the alpha-subunit of the acetylcholine receptor from Torpedo californica

We have studied alkylation of the acetylcholine receptor by N-[3H]ethylmaleimide ([3H]NEM) under various conditions. The radiolabeled preparations were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the receptor complex into subunits, and the incorporation of 3H into each type of chain was determined. We found the following: (i) When cysteines of native receptor in intact membranes were reacted with [3H]NEM, only the beta-subunit was labeled; the extent of alkylation did not change significantly if cholinergic effectors were present during this reaction. (ii) When the disulfide bonds of the receptor were reduced with dithiothreitol (DTT), the alpha- and beta-chains were labeled with [3H]NEM. The presence of receptor agonists and competitive antagonists during alkylation significantly altered the labeling patterns. Gallamine and hexamethonium markedly enhanced, while carbamylcholine and decamethonium markedly lessened, labeling of the alpha-subunit. Choline, d-tubocurarine, and alpha-neurotoxin induced small, but significant decreases in alkylation of the alpha-subunit, while procaine had no effect. (iii) When the same ligands were present during the reduction step, subsequent labeling with [3H]NEM produced patterns similar to those described in (ii). We also investigated the effects of gallamine and hexamethonium on reduction of the disulfide bond located near the acetylcholine binding site by using the affinity alkylating reagent (bromoacetyl)choline (BAC). Gallamine (0.1 mM) was able to increase the rate of reduction of this particular disulfide bond 3-fold in comparison to the control. In these experiments, alkylation by BAC blocked 50% of the toxin binding sites. Hexamethonium (1 mM) had a similar effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity.

Disulfide bonds were found to link the nonglycosylated envelope protein VP-2/M (19 kDa), encoded by open reading frame 6, and the major envelope glycoprotein VP-3 (25 to 42 kDa), encoded by open reading frame 5, of lactate dehydrogenase-elevating virus (LDV). The two proteins comigrated in a complex of 45 to 55 kDa when the virion proteins were electrophoresed under nonreducing conditions but dissociated under reducing conditions. Furthermore, VP-2/M was quantitatively precipitated along with VP-3 in this complex by three neutralizing monoclonal antibodies to VP-3. The infectivity of LDV was rapidly and irreversibly lost during incubation with 5 to 10 mM dithiothreitol (> 99% in 6 h at room temperature), which is known to reduce disulfide bonds. LDV inactivation correlated with dissociation of VP-2/M and VP-3. The results suggest that disulfide bonds between VP-2/M and VP-3 are important for LDV infectivity. Hydrophobic moment analyses of the predicted proteins suggest that VP-2/M and VP-3 both possess three adjacent transmembrane segments and only very short ectodomains (10 and 32 amino acids, respectively) with one and two cysteines, respectively. Inactivation of LDV by dithiothreitol and dissociation of the two envelope proteins were not associated with alterations in LDV's density or sedimentation coefficient.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Selective covalent labeling of cysteines in bovine serum albumin and in hepatoma tissue culture cell glucocorticoid receptors by dexamethasone 21-mesylate

The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.