Steroid binding activity is retained in a 16-kDa fragment of the steroid binding domain of rat glucocorticoid receptors

The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites.     

ATP-dependent activation of L cell glucocorticoid receptors to the steroid binding form.

The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.     

Computer-based search for steroid and DNA binding sites on estrogen and glucocorticoid receptors

The primary amino acid sequences of proteins that are receptors for estrogen, glucocorticoids, and ouabain were compared with each other using computer programs designed to detect and quantify similarities between proteins. Three regions of similarity between the estrogen receptor (ER) and the glucocorticoid receptor (GR) were identified. On the ER, residues 173-250, 323-395, and 426-458 are similar to residues 409-486, 540-612, and 644-676, respectively, on the GR. The ALIGN computer analysis of these segments on the ER and the GR gave comparison scores that were 16.8, 13.7, and 6.8 standard deviations higher, respectively, than that obtained with a comparison of randomized sequences of these proteins. The probability of getting these scores by chance is less than 10(-60), 10(-40), and 10(-11), respectively. Others have proposed that the segment on the ER and GR that is nearest their amino terminus (e.g. residues 173-250 of the ER) is part of their DNA binding domain and that the other two similar segments on each receptor, which are closer to their carboxy terminus, are part of their steroid binding domain. Here, we present evidence to support both of these hypotheses. First, an Align computer analysis indicates that residues 323-395 of the ER and residues 570-612 of the GR contain a region that is similar to a part of the alpha-subunit of the (Na+ + K+)ATPase that is hypothesized to bind the steroid ouabain. This similarity provides additional support for the proposed location of the steroid binding site on the ER, GR, and (Na+ + K+)ATPase. Second, a computer search of the protein sequence database revealed that protamine, a DNA binding protein, has some similarity to residues 255-281 of the ER, which are thought to be part of the DNA binding domain in the ER. Further, we find that residues 276-281 of the ER contain a structure that has been found at the nucleotide binding domain of some protein kinases. If this region on the ER binds ATP, then it may be involved in phosphorylation/dephosphorylation of the ER, which is thought to be important in its mechanism of action.     

Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors

Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA-cellulose using a modification of a protocol developed for purification of rat GCR. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species.     

Corepressor binding to progesterone and glucocorticoid receptors involves the activation function-1 domain and is inhibited by molybdate

Corepressors are known to interact via their receptor interaction domains (RIDs) with the ligand binding domain in the carboxyl terminal half of steroid/nuclear receptors. We now report that a portion of the activation function-1 domain of glucocorticoid receptors (GRs) and progesterone receptors (PRs), which is the major transactivation sequence, is necessary but not sufficient for corepressor [nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT)] RID binding to GRs and PRs in both mammalian two-hybrid and coimmunoprecipitation assays. Importantly, these two receptor sequences are functionally interchangeable in the context of GR for transactivation, corepressor binding, and corepressor modulatory activity assays. This suggests that corepressors may act in part by physically blocking portions of receptor activation function-1 domains. However, differences exist in corepressor binding to GRs and PRs. The C-terminal domain of PRs has a higher affinity for corepressor than that of GRs. The ability of some segments of the coactivator TIF2 to competitively inhibit corepressor binding to receptors is different for GRs and PRs. With each receptor, the cell-free binding of corepressors to ligand-free receptor is prevented by sodium molybdate, which is a well-known inhibitor of receptor activation to the DNA-binding state. This suggests that receptor activation precedes binding to corepressors. Collectively, these results indicate that corepressor binding to GRs and PRs involve both N- and C-terminal sequences of activated receptors but differ in ways that may contribute to the unique biological responses of each receptor in intact cells.     

Protein-protein interactions facilitate DNA binding by the glucocorticoid receptor DNA-binding domain

We have studied the interaction of the DNA-binding domain of the glucocorticoid receptor with a glucocorticoid response element from the tyrosine aminotransferase gene. This response element consists of two binding sites (half-sites) for the glucocorticoid receptor DNA-binding domain. The sequences of these two half-sites are not identical, and we have previously shown that binding occurs preferentially to one of the half-sites (Tsai, S.-Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson, J.-A., Tsai, M.-J., and O'Malley, B. W. (1988) Cell 55, 361-369). We show here that binding to the low affinity half-site is dependent on previous occupancy of the high affinity half-site. This facilitated binding is dependent on the distance between the two half-sites and their relative orientation but is not dependent on the integrity of the DNA backbone. This is consistent with a model where DNA binding is not only dependent on interactions between the protein and its DNA target sequence but is also influenced by interactions between the protein molecules bound.     

Thermodynamics of steroid binding to the human glucocorticoid receptor.

The thermodynamics of the interaction of glucocorticoids with their receptor were studied in cytosol from human lymphoblastoid cells. The rate and affinity constants of dexamethasone and cortisol between 0 degree and 25 degrees C were calculated by curve-fitting from time-course and equilibrium kinetics. The data were consistent with a simple reversible bimolecular interaction. Arrhenius and Van't Hoff plots were curvilinear for both steroids. At equilibrium, the solution for the equation delta G = delta H - T X delta S (eqn. 1) was (in kJ X mol-1) -47 = 36 - 83 (dexamethasone) and -42 = -9 - 33 (cortisol) at 0 degree C. Enthalpy and entropy changes decreased quasi-linearly with temperature such that, at 25 degrees C, the respective values were -50 = -75 + 25 and -43 = -48 + 5. Thus, for both steroids, the interaction was entropy-driven at low temperature and became entirely enthalpy-driven at 20 degrees C. Thermodynamic values for the transition state were calculated from the rate constants. For the forward reaction, eqn. (1) gave 45 = 84 - 39 (dexamethasone) and 46 = 60 - 14 (cortisol) at 0 degree C, and 44 = 24 + 20 (dexamethasone) and 46 = 28 + 18 (cortisol) at 25 degrees C. These data fit quite well with a two-step model [Ross & Subramanian (1981) Biochemistry 20, 3096-3102] proposed for ligand-protein interactions, which involves a partial immobilization of the reacting species governed by hydrophobic forces, followed by stabilization of the complex by short-range interactions. On the basis of this model, an analysis of the transition-state thermodynamics led to the conclusion that no more than half of the steroid molecular area is engaged in the binding process.     

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

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

The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.

Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.     

Association of heat shock protein 90 with the 16 kDa steroid binding core fragment of rat glucocorticoid receptors

We have recently described a 16 kDa steroid binding core (Thr537-Arg673) of the rat glucocorticoid receptor [Simons et al. (1989) J. Biol. Chem. 264, 14493-14497]. Sedimentation analysis and size exclusion and anion exchange chromatography now suggest that other proteins are associated with the 16 kDa receptor, just as has been seen for the intact 98 kDa receptor. The 16 kDa fragment was also immunoprecipitable with anti-heat shock protein 90 (hsp90) antibody. These results argue that hsp90 binds to the 16 kDa core fragment and directly position the site of hsp90 association between Thr537 and Arg673 of the rat glucocorticoid receptor.     

Preferential inhibition of the oncogenic form of RasH by mutations in the GAP binding/"effector" domain

The double mutation, D33H/P34S, reduced the transforming activity of oncogenic RasH proteins, G12V and Q61L, 400- and 20-fold, respectively. Remarkably, this same mutation did not reduce the transforming activity of normal RasH, nor did it impair the ability of the protein to restore a functional Ras pathway in cells whose endogenous Ras proteins were inhibited. Another mutation in this region, D38N, had similar effects. The mutations reduced downstream coupling efficiency of normal Ras as assessed by yeast adenylyl cyclase stimulation. However, this was offset by decreased GTPase activating protein (GAP) binding, since the latter resulted in elevated GTP-bound mutant Ras in cells. The mutations produced a similar decrease in downstream coupling efficiency of oncogenic Ras, but decreased GAP binding did not compensate because the GTPase activity of oncogenic Ras is not stimulated by GAP. These results imply that preferential inactivation of oncogenic Ras in human tumors may be achieved by reagents designed to inhibit the GAP-binding/"effector" domain of Ras proteins.     

Analysis of the steroid binding domain of rat steroid 5alpha-reductase (isozyme-1): the steroid D-ring binding domain of 5alpha-reductase.

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5alpha-androst an-17beta-carboxamide is an effective probe of rat steroid 5alpha-reductase (isozyme-1) (5alphaR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5alphaR-1 activity were ultraviolet (UV)-photolyzed with [3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5alphaR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55-56 min. Rechromatography of this fraction using a modified gradient (elution 54-55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15-18 of the 5alphaR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an approximately 12-fold increase in the Km for testosterone, whereas the Km for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.     

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.