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.     

Analysis of the glucocorticoid antagonist action of dexamethasone 21-mesylate in HeLa S3 cells

Several properties of human glucocorticoid receptors complexed to the synthetic glucocorticoid agonists dexamethasone (DEX) and triamcinolone acetonide (TA) and the antagonist dexamethasone 21-mesylate (DM) are compared in an attempt to define the mode of action of DM. Both DEX and TA induce an increase in alkaline phosphatase activity in HeLa S3 cells. Not only is DM without effect on alkaline phosphatase activity at concentrations as great as 10(-7) M, it blocks the action of DEX and TA on enzyme induction, thus acting as a pure antagonist in this system. DM-receptor complexes, like agonist-receptor complexes, are recovered in the cytosol when cells are incubated with ligand at 0 degrees C but are recovered from the nucleus when incubation is shifted to 37 degrees C, suggesting that activation of the antagonist-receptor complex occurs in vivo. The molecular species that undergoes this temperature-dependent shift from the cytosolic compartment to the nuclear compartment exhibits saturable binding to the antagonist. Both the cytosolic and nuclear species exhibit a relative molecular mass of approximately equal to 94,000 Daltons when analysed by SDS-polyacrylamide gel electrophoresis. Receptors labeled in intact cells with [3H]DM at 0 degrees C sediment at approximately 8S in sucrose gradients, shifting to 4S when the gradients contain 0.4 M KCl. DEX- and TA-labeled receptors show the same sedimentation behavior, which has been accepted as one criterion of receptor subunit dissociation, or activation.     

Identification of cysteine 656 as the amino acid of hepatoma tissue culture cell glucocorticoid receptors that is covalently labeled by dexamethasone 21-mesylate

Recent results using proteases suggest that dexamethasone 21-mesylate (Dex-Mes) labeling of the rat hepatoma tissue culture (HTC) cell glucocorticoid receptor occurs at one or a few closely grouped cysteine residues (Simons, S.S., Jr. (1987) J. Biol. Chem. 262, 9669-9675). In this study, a more direct approach was used both to establish that only one cysteine is labeled by [3H]Dex-Mes and to identify the amino acid sequence containing this labeled cysteine. Various analytical procedures did not provide the purification of the extremely hydrophobic Staphylococcus aureus V8 protease digestion fragment that is required for unique amino acid sequencing data. Therefore, Edman degradation was performed on the limit protease digest mixtures which appeared to contain only one 3H-labeled peptide. These degradation experiments revealed the number of amino acid residues between the NH2 terminus of each peptide and the [3H]Dex-Mes-labeled cysteine. A comparison of these amino acid spacings with the published amino acid sequence of the HTC cell glucocorticoid receptor (Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikstom, A-C., Gustafsson, J-A., and Yamamoto, K. R. (1986) Cell 46, 389-399) indicated that the one cysteine labeled by [3H]Dex-Mes is Cys-656. Further analysis of the receptor sequence for the presence of the observed grouping of proteolytic cleavage sites, but without any preconditions as to which amino acid was labeled, gave Asp-122 and Cys-656 as the only two possibilities. Potential labeling of Asp-122 could be eliminated on the basis of immunological and genetic evidence. We, therefore, conclude that the single Dex-Mes-labeled site of the HTC cell glucocorticoid receptor has been identified as Cys-656. Since several lines of evidence indicate that [3H]Dex-Mes labeling of the receptor occurs in the steroid binding site, Cys-656 is the first amino acid which can be directly associated with a particular property of the glucocorticoid receptor.     

Characterization of glucocorticoid receptors in S49 mouse lymphoma cells by affinity labeling with [3H]dexamethasone 21-mesylate

Glucocorticoid receptors in wild type and mutant S49 mouse lymphoma cells were affinity labeled with [3H]dexamethasone 21-mesylate and analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of receptors in cytosol from wild type cells and nuclear transfer decreased (nt-) mutants was 97,000 (97 kDa). The molecular weight of receptors in cytosol from nuclear transfer increased (nti) mutants was 48 kDa. The 97 kDa receptor in cytosol from wild type cells was digested by chymotrypsin to a 40 kDa steroid-binding receptor fragment but the 48 kDa receptor in cytosol from nti mutants was resistant to digestion by chymotrypsin. In addition to the 48 kDa receptor, cytosol from nti mutants contained 40 and 18 kDa receptor fragments. Cytosol from the nt- mutants also contained 18 kDa receptor fragments. The 40 and 18 kDa receptor fragments were present in multiple subclones of a nti mutant cell line. Formation of these receptor fragments was not prevented by protease inhibitors and was not increased by extended incubation of cytosol samples. Both 48 and 40 kDa forms of the receptor, but not the 18 kDa form, could be activated and bound by DNA-cellulose.     

High-resolution anion exchange chromatography of the glucocorticoid receptor from WEHI-7 cells

A simple refinement of the current methods of DEAE chromatography of steroid receptors has been developed which takes advantage of the characteristics of Fast Flow DEAE Sepharose (Pharmacia). The approach provides a convenient and inexpensive means to carry out high-resolution chromatography of the glucocorticoid receptor. Using this method, at least five separate species of receptor have been detected within the single so-called "low-salt" peak normally seen using the current methods of receptor anion exchange chromatography.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

Identification by site-directed mutagenesis of Lys-558 as the covalent attachment site of H2DIDS in the mouse erythroid band 3 protein

After functional expression of mouse erythroid band 3 by cRNA microinjection into Xenopus oocytes, 36Cl- efflux is irreversibly inhibited by H2DIDS. When a cRNA is injected that is derived from a cDNA in which the nucleotides encoding for lysine-558 were replaced by nucleotides encoding for asparagine, transport and inhibition of transport by H2DIDS still occur. However, when measured under conditions where no intramolecular crosslinking takes place the inhibition by H2DIDS is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 takes place at Lys-558.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Mechanisms of glucocorticoid function in human leukemic cells: analysis of receptor gene mutants of the activation-labile type using the covalent affinity ligand dexamethasone mesylate

In the cultured acute lymphoblastic leukemic (ALL) cell line, clones of sensitive cells are killed by receptor-occupying concentrations of glucocorticoids. In addition, several types of resistance have been identified. The types of resistance are r- (glucocorticoid binding site loss), ract/l (activation labile receptors) and r+ly- (defective lysis mechanism). The two types of receptor mutants have been examined for the presence and expression of the glucocorticoid receptor (GR) gene. Southern blot analysis, using a full-length cDNA probe for human GR, shows that the gene in both is grossly intact. Examination of the expression of the gene by Northern blots reveals the presence of normal, 7-kb message in both types of receptor mutants, though in amounts somewhat reduced from wild-type. This report focuses on the activation labile mutants. Since characterization of these mutants suggests that they can bind ligand but not retain it during activation, we hypothesized that they would respond normally to a ligand that could not be lost during activation. This seems to be the case. When the covalent affinity ligand dexamethasone mesylate, itself a partial glucocorticoid agonist/antagonist, is used, the ract/l cells are killed to an extent corresponding to that evoked by a sub-optimal concentration of the full agonist dexamethasone. We conclude: (1) that the ract/l receptors can function to kill cells if provided a ligand that they do not lose during activation; (2) that the partial agonist activity of dexamethasone mesylate for cell killing is not due to release of a small amount of free dexamethasone; (3) that the poor agonist activity of dexamethasone mesylate receptor complexes suggests that the role of steroid is strictly to participate in conversion of the receptor to its DNA binding form, after which presence of the steroid actually interferes with proper receptor action.     

Methionine 500, the site of covalent attachment of an active site-directed reagent of beta-galactosidase.

The site of attachment to beta-galactosidase of the active site-directed inhibitor, beta-D-galactopyranosylmethyl p-nitrophenyl triazene, was determined. When the enzyme is completely inactivated, 1 mol of the galactopyranosylmethyl group is bound per mol of monomer with retention of the tetrameric structure. After reaction with the [14C]methyl reagent, labeled peptides were isolated and analyzed. The radioactive label was found to be covalently bound to methionine residue 500.     

Transfection of cDNA with G----T point mutation at the cleavage site of insulin receptors to COS 7 cells

To study whether the G----T point mutation of insulin proreceptors at the cleavage site which changed -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser- caused unprocessed insulin receptors with decreased insulin binding affinity, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. After site-directed mutagenesis, an expression vector pGEM3SV was used to make a plasmid which contained full-length HIRcDNA behind SV40 early promoter. Transfection of normal HIR cDNA produced normal insulin receptors on the plasma membranes in COS 7 cells. However, transfection of cDNA with the mutation resulted in the presence of 210K proreceptors in the plasma membranes with decreased insulin binding ability (35% of normal). These results suggest that the mutation, not the defect of converting enzyme, was the cause for unprocessed insulin proreceptors in the patients with insulin resistance.     

Kinetics of dexamethasone binding to the glucocorticoid receptor of intact erythroid cells from rat fetal liver

The erythroid cells from the rat fetal liver have been shown to possess a receptor for glucocorticoids. In the present work, the characteristics of [3H]dexamethasone binding have been studied on intact cells, in order to minimize receptor degradation, and at 4 degrees C, in order to prevent the activation of the hormone-receptor complex. Dissociation kinetics were those of a first-order reaction and the value of the rate constant of dissociation was similar to the values available in the literature. When studied at low concentrations of the ligand and using short-term incubations, association kinetics were apparently those of a simple bimolecular reaction. But at high ligand concentrations and/or using long-term incubations, association kinetics indicated a more complex reaction. Our results were compatible with the model proposed by Pratt W.B., Kaine J.L. and Pratt V.D. (J. Biol. Chem. 250 (1975) 4584-4591) for cytosolic preparations. This model implies the rapid formation of a transient unstable form of the complex, further converted into a stable form with slower kinetics. Equilibrium dissociation constant of the first (rapid) reaction was 80 microM and the rate constant of 'stabilization' was of the order of 70 X 10(-3) min-1. These values agree with the results of Pratt et al. relative to a cytosolic preparation from rat thymocytes.     

Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein

In order to determine the ratio of phosphates to hormone-binding sites on nonactivated (non-DNA-binding) glucocorticoid receptors in WEHI-7 mouse thymoma cells, we have extracted these receptors from cells grown to a steady state with 32P, labeled them with a saturating concentration of [3H]dexamethasone 21-mesylate, purified them using a monoclonal antibody, and analyzed them by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The complexes contained approximately 5 mol of phosphate/mol of bound steroid. Only half of the phosphates were associated with the approximately 100-kDa protein which is labeled with [3H]dexamethasone 21-mesylate. The remaining phosphates were associated with the approximately 90-kDa non-steroid-binding component of the nonactivated complex. Dual label studies, using [35S]methionine to measure receptor protein and 32P to measure receptor phosphates, have enabled us to determine the phosphate content, relative to receptor protein, of both nonactivated and activated cytosolic complexes generated in intact WEHI-7 cells exposed to triamcinolone acetonide at 37 degrees C. The total amount of phosphate associated with the activated complex is roughly half of that associated with the nonactivated complex, the decrease being accounted for by dissociation of the approximately 90-kDa phosphoprotein which accompanies activation. However, the ratio of 32P to 35S counts associated with the approximately 100-kDa steroid-binding protein is the same for the activated and nonactivated complexes. These results indicate that there is no net change in the phosphorylation of the approximately 100-kDa steroid-binding component of the cytosolic glucocorticoid-receptor complex upon activation in the intact cell.     

Poly(ethylene glycol)-supported enzyme inactivators. Efficient identification of the site of covalent attachment to alpha-chymotrypsin by PEG-TPCK

A new methodology utilizing an enzyme inactivator covalently attached to poly(ethylene glycol) (PEG) is described in which the PEG affords facile and mild quantification, isolation, and identification of the site of enzyme inactivation. As proof of concept, the known affinity labeling agent for alpha-chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), was linked to PEG. The synthesis of the PEG-bound inactivator PEG-TPCK was carried out in good yields using standard solution-phase chemistry. Inactivation of alpha-chymotrypsin with PEG-TPCK was monitored via UV-vis spectroscopy in aqueous conditions, which resulted in less than 3% remaining activity, indicating that 97% of the alpha-chymotrypsin was covalently modified with PEG-TPCK. The MALDI-TOF mass spectrum showed only one new peak that was distinct in shape and corresponded to the mass of PEG-TPCK-alpha-chymotrypsin. Following proteolytic digestion, the PEG-TPCK-peptide was easily discernible from the rest of the digest in a HPLC trace because of its characteristic prolonged retention time and broad polymer shape. MALDI-TOF MS was used to determine the mass of the PEGylated peptide. Without prior removal of the PEG, the amino acid site to which PEG-TPCK covalently bound was determined via Edman sequencing. In comparison to other methods, the PEG-supported inactivator system is significantly cheaper and safer than the synthesis of radiolabeled compounds; furthermore, isolation of the PEGylated peptide is milder and more selective than standard affinity binding columns. Edman sequencing provides an exact determination of the site of inactivator covalent attachment without extensive, tedious LC-MS analysis of a complex peptide mixture. The method described here could be applied to a variety of enzymes as an alternative to current techniques.     

Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABA_A receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABA_A receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [³H]FNZP binding.     

Biochemical analysis of Hyphantria cunea NPV attachment to Spodoptera frugiperda 21 cells

Binding characteristics of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) to Spodoptera frugiperda 21 (Sf21) cells was determined. The cells displayed an affinity of 0.9 × 10¹⁰ M^-1 with about 8900 binding sites per cell. The biochemical nature of HcNPV-binding sites on the cell surface was also partially elucidated. There were 45 to 49% reductions in HcNPV binding following the pretreatment of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, which inhibits N-linked glycosylation and the expression of some membrane proteins on the cell surface, reduced virus binding suggesting a role for glycoprotein(s) in binding. Treatment of cells with wheat germ agglutinin or neuraminidase did not measurably reduce virus binding, indicating that oligosaccharides containing N-acetylglucosamine or sialic acid are not directly involved in HcNPV attachment. The negative effect of methylamine on HcNPV binding seems to be due to the fact that HcNPV entry via an endocytic pathway is blocked by the increased pH of the endosome. Data on energy inhibitors (sodium azide and dinitrophenol) indicates that HcNPV attachment to Sf21 cells may be closely linked to viral entry via receptor-mediated endocytosis. These findings suggest that the binding site moiety has a glycoprotein component, but that direct involvement of oligosacccharides containing N-acetylglucosamine or sialic acid residues in binding is unlikely, and that HcNPV attachment to Sf21 cells might be via receptor-mediated endocytosis. This revised version was published online in August 2006 with corrections to the Cover Date.