Steroid Binding Capacity Research Articles

N-Glycosylation influences human corticosteroid-binding globulin measurements.

ObjectiveDiscrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG.Methods and subjectsMolecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects.ResultsWe demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients.ConclusionsPlasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma.

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

Functional implications of corticosteroid-binding globulin N-glycosylation.

Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2–4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.

Endocrine Modulation in Atlantic Cod (Gadus morhua L.) Exposed to Alkylphenols, Polyaromatic Hydrocarbons, Produced Water, and Dispersed Oil

Effluent from oil production activities contains chemicals that are suspected of inducing endocrine disruption in fish. In this study, Atlantic cod (Gadus morhua L.) were exposed to mixtures of low- and medium-molecular-weight alkylphenols (AP) (methyl- to heptylphenol), polycyclic aromatic hydrocarbons (PAH), diluted produced water, and dispersed oil for 15 d in a flow-through exposure system. Condition index (CI), hepatosomatic index (HSI), gonadosomatic index (GSI), concentration of the estrogenic biomarker vitellogenin (Vtg), and modulation of the total sex steroid-binding capacity in plasma were determined to assess whether these mixtures were capable of interfering with endocrine-regulated physiological processes in Atlantic cod. No marked differences in plasma Vtg levels were found between control and exposed groups of either males or females, possibly due to high intergroup variances and low sample numbers. An apparent numerical increase in the number of male and female fish with high plasma Vtg levels was, however, observed in some exposure groups compared to control. This purported weak estrogenic effect was several orders of magnitude lower than that observed for potent estrogens and suggested that the levels of estrogen receptor (ER) agonists were low. Exposure of female fish to a mixture of dispersed oil and a mixture of AP, PAH, and dispersed oil led to upregulation of the plasma total sex steroid-binding capacity, indicating interference with the normal blood steroid transport. No significant effects were seen for CI, HSI, and GSI, suggesting that the endocrine-disrupting potential was not sufficient to elicit effects on general physiological conditions and gonad development during this short exposure period.

Steroid binding properties of the 2nd WHO International Standard for sex hormone-binding globulin

Stocks of 95/560, the 1st International Standard for sex hormone-binding globulin (SHBG), are running out, and 08/266 has been prepared as a replacement. We compared the steroid binding capacities of 95/560 and 08/266. 95/560 and 08/266 stored at -20 °C, and accelerated thermal degradation samples of 08/266 were subjected to gelfiltration. Binding of the SHBG-containing fractions to a dihydrotestosterone derivative was then investigated using a surface plasmon resonance biosensor. The SHBG content of the gelfiltration fractions was determined with an immunoassay. Steroid binding capacity of each standard was then calculated as the mean of the ratio of biosensor binding/SHBG concentration. Affinity constants for the SHBG steroid interaction were calculated. Maximum achievable biomolecular interactions were lower for 95/560 than for the 08/266 preparations. 95/560 exhibited steroid binding capacity only half as high as any of the novel standards, as well as a lower affinity constant for the SHBG steroid interaction. No significant differences could be observed between the samples of 08/266 stored at different temperatures. The steroid binding capacity of 08/266 is two times higher than that of 95/560. Steroid binding of lyophilized 08/266 is preserved at storage temperatures of up to 45 °C for at least 6 months.

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

Dietary restriction reduces the rate of estradiol clearance in sheep (Ovis aries)

Three experiments were designed to test the effect of dietary restriction on clearance of 17beta-estradiol (E(2)) in sheep. A preliminary experiment examined the effect of a 4-d fast on the rate of E(2) clearance in wethers. The second experiment tested the hypothesis that either long-term restriction (7 wk) or a 5-d fast would increase steroid-binding capacity of serum by increasing the concentration of sex hormone-binding globulin (SHBG) in the blood of ovariectomized ewes. In Exp. 3, we hypothesized that nutrition-dependent regulation of E(2) clearance by the liver would result in divergence in biliary extraction of E(2) in fed and fasted wethers receiving comparable levels of exogenous E(2). A marked difference in E(2) clearance between fed and fasted wethers was noted in the preliminary study. Relative to ad libitumfed wethers, a 4-d fast decreased E(2) clearance by 52%. Serum concentrations of SHBG were increased in long-term energy-restricted and fasted ewes, relative to the concentration in maintenancefed ewes (P = 0.015). Furthermore, a 5-d fast nearly doubled serum steroid-binding capacity in wethers. The E(2) concentration in bile was 2 times greater in fasted than in fed wethers. This fasting-dependent increase in biliary E(2) may be reflective of the increased serum E(2) in fasted animals, because each 1 pg/mL increase in serum E(2) increased bile E(2) by 0.86 +/- 0.12 pg/mL, independent of nutrition (P = 0.002). Our results demonstrate that the rate of clearance of E(2) is decreased during nutritional restriction. Additionally, these data indicate that altered SHBG expression, enterohepatic recirculation, or both are involved in the decreased E(2) clearance during dietary restriction.

Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials ( Phascogale tapoatafa) that survive beyond the breeding season in captivity

The semelparous dasyurids display a unique life history, in that all males die within a few weeks of the completion of the breeding season. Studies of several semelparous species have revealed that the male die-off is stress-related, and accompanied by increased plasma androgen and cortisol levels and decreased corticosteroid binding capacity, resulting in suppression of immune and inflammatory responses. This study examines the endocrine profile of male brush-tailed phascogales ( Phascogale tapoatafa) that survive beyond the breeding season in captivity. Plasma cortisol, corticosteroid binding globulin and albumin levels were monitored in both males and females and steroid partitioning calculated. Captive males surviving beyond the breeding season did not show the elevation in plasma cortisol and decrease in corticosteroid binding capacity reported in wild males. Plasma albumin concentrations also remained constant during the sampling period. These data indicate that captive males do not undergo the same stress response described in wild populations.

Effect of undernutrition on uterine progesterone and oestrogen receptors and on endocrine profiles during the ovine oestrous cycle

In the present study, it was investigated whether undernutrition affected the binding capacity, immunoreactivity and mRNA expression for uterine oestrogen and progesterone receptors (ER and PR, respectively) in sheep, as well as whether the responses were associated with changes in plasma concentrations of progesterone (P4), oestradiol (E2), glucose, fatty acids, insulin, leptin and insulin-like growth factor (IGF)-I during the oestrous cycle. Twenty ewes were fed either 1.5 (C) or 0.5 (L) times their maintenance requirements and were killed on Day 5 or 14 of the cycle (Day 0 = oestrus). Compared with Group C, Group L had higher concentrations of non-esterified fatty acids and lower concentrations of insulin, leptin and IGF-I. Group L also had higher plasma concentrations of P4 during the final days of the luteal phase. At oestrus in both treatment groups, there were peaks in the concentrations of glucose, insulin and IGF-I. For ER and PR, transcript expression, binding capacity and immunoreactivity were higher on Day 5 than on Day 14 of the cycle. The binding capacities for ER and PR were lower in Group L than in Group C on Day 5. Group C showed more immunoreactive staining for ER than did Group L in two of five cell types, whereas no effect of treatment was observed for PR immunoreactivity. There was more PR mRNA in the uterine horn contralateral to the corpus luteum in Group C than in Group L ewes. We conclude that undernutrition impairs steroid receptor expression and binding capacity. This may alter the uterine environment and help explain the reductions in embryo survival.

Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.

Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation.

A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220-amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants.

Glucocorticoid receptor nitration leads to enhanced anti-inflammatory effects of novel steroid ligands.

It has recently emerged that posttranslational modification of proteins via nitration of tyrosine residues can alter their function. In this study, we describe that specific nitration of the glucocorticoid receptor (GR) by NCX-1015, a novel NO-donating prednisolone derivative (prednisolone 21-[4'-(nitrooxymethyl)benzoate), results in an enhancement of GR-mediated events. Incubation of PBMC and U937 cells with 1-10 micro M NCX-1015 caused faster activation of GR as assessed by augmented 1) binding to [(3)H]dexamethasone, 2) dissociation from heat shock protein 90, and 3) nuclear translocation. PBMCs treated with NCX-1015 contained GR that had undergone tyrosine nitration. The chemistry facilitating the increase in steroid binding capacity observed with NCX-1015 is specific, because changing the position of the NO-donating group or ubiquitous nitration by addition of an NO donor was unable to mimic this event. In vivo treatment with NCX-1015 provoked GR nitration and faster heat shock protein 90 dissociation as assessed in peritoneal cells. Accordingly, NCX-1015, but not prednisolone or other derivatives, produced a rapid inhibition of the early neutrophil recruitment and mediator generation in a model of peritonitis. In conclusion, we report here for the first time that posttranslational modification of GR by this novel nitrosteroid is associated with its enhanced anti-inflammatory activity.

Mutations at Positions 547–553 of Rat Glucocorticoid Receptors Reveal That hsp90 Binding Requires the Presence, but Not Defined Composition, of a Seven-amino Acid Sequence at the Amino Terminus of the Ligand Binding Domain

Glucocorticoid receptors (GRs) must heterocomplex with hsp90 to have an open steroid binding cleft that can be accessed by steroid. We reported that a seven-amino acid sequence (547-553 of rat GR) overlapping the amino-terminal end of the ligand binding domain is required for hsp90 binding to GR. We have now conducted saturation mutagenesis of this sequence, which appears to be part of the surface where the ligand binding cleft merges with the surface of the ligand binding domain. No single point mutation causes significant changes in any of a variety of biochemical and biological properties in addition to hsp90 binding. A triple mutation (P548A/T549A/V551A) increases by >100-fold the steroid concentration required for half-maximal induction without affecting the level of maximal induction or coactivator response. Interestingly, this triple mutant displays reduced binding of steroid and hsp90 in whole cells, but it possesses wild type affinity for steroid and normal hsp90 binding capacity under cell-free conditions. This phenotype of a dramatic shift in the dose response for transactivation would be expected from an increase in the rate of disassembly of the triple mutant GR.hsp90 heterocomplex in the cell. Mutation of the entire seven-amino acid region to CAAAAAC maintains the presence of a critical alpha-helical structure and heterocomplex formation with hsp90 but eliminates steroid binding and transcriptional activation, thus disconnecting hsp90 binding from opening of the ligand binding cleft and steroid binding.

Modification of an essential amino group in the mineralocorticoid receptor evidences a differential conformational change of the receptor protein upon binding of antagonists, natural agonists and the synthetic agonist 11,19-oxidoprogesterone

The alkylation of amino groups of the mineralocorticoid receptor (MR) with pyridoxal 5′-phosphate or 2,4,6-trinitrobenzenesulphonate (TNBS) under controlled conditions modifies only one lysyl residue, which accounts for a 70% inhibition of steroid binding capacity. The K d of aldosterone for MR is not affected by the treatment, but the total number of binding sites is greatly decreased. The modified receptor is capable of dynamically conserving its association with the hsp90-based heterocomplex. Importantly, the binding of natural agonists protects the hormone binding capacity of the MR from the inactivating action of alkylating agents. In contrast, antagonistic steroids are totally incapable of providing such protection. Like the antagonistic ligands, and despite its potent mineralocorticoid biological effect, the sole MR specific synthetic agonist known to date, 11,19-oxidoprogesterone (11-OP), shows no protective effect upon treatment of the MR with pyridoxal 5′-phosphate or TNBS. Limited digestion of the MR with α-chymotrypsin generates a 34 kDa fragment, which becomes totally resistant to digestion upon binding of natural agonists, but not upon binding of antagonists. Interestingly, the synthetic 21-deoxypregnanesteroid 11-OP exhibits an intermediate pattern of proteolytic degradation, suggesting that the conformational change generated in the MR is not equivalent to that induced by antagonists or natural agonists. We conclude that in the first steps of activation, the MR changes its conformation upon binding of the ligand. However, the nature of this conformational change depends on the nature of the ligand. The experimental evidence shown in this work suggests that a single lysyl group can determine the hormone specificity of the MR.

Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer.

Human sex hormone-binding globulin (SHBG) transports sex steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its steroid binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single steroid-binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and steroid-binding sites are distinct and that both monomers within a homodimeric complex are capable of binding steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a steroid-binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical steroid binding capacities. These data indicate that both subunits of the SHBG homodimer bind steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket.

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.

Oxidative stress induced by L-buthionine-( S, R)-sulfoximine, a selective inhibitor of glutathione metabolism, abrogates mouse kidney mineralocorticoid receptor function

In vitro studies have demonstrated that cysteine groups present in most of the steroid receptors play an essential role in the steroid binding process as well as in the ability of this superfamily of signaling proteins to function as transcription factors. However, there is poor experimental evidence, if any, which demonstrates that under conditions of oxidative stress the steroid receptors in general, and the mineralocorticoid receptor in particular, are affected in vivo in a similar fashion as has been described for cell-free systems or cells in culture. In the present work we report that when mice are exposed to oxidative stress by treatment with L-buthionine-( S, R)-sulfoximine ( L-( S, R)-BSO), a glutathione depleting agent, the aldosterone-dependent mineralocorticoid biological response (measured as sodium retention and potassium elimination) was diminished in a directly proportional manner with respect to the depletion of renal glutathione. Accordingly, the steroid binding capacity of the mineralocorticoid receptor was also abrogated, whereas the receptor protein level remained unchanged. The harmful effects observed in mice after glutathione depletion were efficiently prevented by co-treatment with glutathione monoethyl ester. Similar inhibition in the steroid binding capacity was also generated in vitro by receptor alkylation and receptor oxidation, an effect which was prevented in the presence of reducing agents. Since the glutathione deficit generated in vivo by treatment with L-( S, R)-BSO did not significantly affect other renal proteins which are known to be required for the mineralocorticoid mechanism of action, we suggest that in renal cells a low redox potential exerts drastic and uncompensated inhibition of the receptor-mediated mineralocorticoid biological response. This effect was ascribed to the loss of steroid binding capacity of oxidized receptor, most likely by modification of essential cysteines as supported by experiments where a decreased number of reactive thiols and reduced covalent binding of thiol-reactive ligand were evidenced on immunopurified receptor after in vivo treatment with L-( S, R)-BSO .

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.

Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex

Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex

Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex

We analysed the inhibitory effects in vitro and in vivo of several metal ions on aldosterone binding to the rat kidney mineralocorticoid receptor with the purpose of assessing possible toxic effects of those ions on sodium retention, as well as to obtain information on receptor structural requirements for ligand binding. For the assays in vitro, the inhibitory effects of 20 metal ions were analysed on steroid-binding capacity for renal receptor cross-linked to 90-kDa heat-shock protein (hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking prevented the artifactual dissociation of hsp90 (and, consequently, the loss of steroid binding) from the mineralocorticoid receptor due to the presence of high concentrations of salt in the incubation medium. Cross-linked heterocomplex showed no difference in ligand specificity and affinity with respect to native receptor, but increased stability upon thermal- or ionic-strength-induced destabilization was observed. Treatments in vitro with metal ions in the range 10(-8)-10(-1) M resulted in a differential inhibitory effect for each particular ion on aldosterone binding. Using the negative logarithm of metal concentration for 50% inhibition, the ions could be correlated with their Klopman hardness constants. The analysis of this relationship led us to postulate three types of reaction: with thiol, imidazole and carboxyl groups. The essential role played by these residues in steroid binding was confirmed by chemical modification of cysteines with dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and acidic amino acids with Woodward's reagent (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic effects of some metal ions were also observed by treatments in vivo of adrenalectomized rats on both steroid-binding capacity and aldosterone-dependent sodium-retaining properties. We suggest that those amino acid residues are involved in the activation process of the mineralocorticoid receptor upon steroid binding. Thus toxic effects observed with these metal ions may be a consequence of modifications of those essential groups. Our results support the notion that toxicity of metals on renal mineralocorticoid function may be predicted according to their chemical hardness.