Selective molybdate-directed covalent modification of sulfhydryl groups in the steroid-binding versus the DNA-binding domain of the glucocorticoid receptor

Hydrogen peroxide produces all of the effects on glucocorticoid receptors that are produced by molybdate, including stabilization of the receptor 90-kDa heat shock protein (hsp90) complex (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). When the glucocorticoid receptor is exposed simultaneously to molybdate and peroxide at concentrations that are optimal for receptor stabilization if each agent is present alone, there is an irreversible loss of steroid binding activity. The effect is accompanied by a covalent modification of the receptor, which is demonstrated by an increase in its apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of the receptor with the sulfhydryl-modifying reagents methyl methanethiosulfonate or N-ethylmaleimide prevents covalent modification, suggesting that cysteine moieties are the site of attack. The covalently modified receptor can still bind to DNA. Molybdate-peroxide treatment does not covalently modify the 15-kDa tryptic fragment containing the DNA-binding domain and 11 of the 20 cysteine moieties in the receptor. However, the 27-kDa tryptic fragment, which contains the steroid-binding domain and 5 cysteines, is covalently modified. The 27-kDa tryptic fragment is covalently modified by the molybdate-peroxide combination when [3H]dexamethasone 21-mesylate is covalently bound to Cys-644. This leaves some combination of 4 cysteines in the steroid-binding domain (628, 649, 671, and 742) as the modified groups. These modifications occur in a region of the receptor that is known to contain its sites of interaction with both hsp90 and molybdate, with the latter having a well-established avidity for sulfur. These observations raise the possibility that the covalent modification caused by the molybdate-peroxide combination represents a modification of sulfur ligands involved in molybdate stabilization of the receptor.     

DNA-binding and non-DNA-binding forms of the transformed glucocorticoid receptor

In this work we have probed the mechanism responsible for two non-DNA-binding states of the mouse glucocorticoid receptor. In the first case, transformed receptors were treated with hydrogen peroxide. It is known that oxidizing agents promote the formation of disulfide bonds in the glucocorticoid receptor, but it has not been determined what domains are involved in any disulfide bond formation that leads to inactivation of DNA-binding activity. We show here that hydrogen peroxide inhibits DNA-binding by the 15-kDa tryptic fragment containing the DNA-binding fingers with the same concentration dependency as it inhibits DNA-binding by the uncleaved receptor. This suggests that all of the effect of peroxide is on sulfhydryl groups within the zinc fingers. After dissociation (transformation) of cytosolic heteromeric glucocorticoid receptor complexes, only a portion (40–60%) of the dissociated receptors can bind to DNA-cellulose. We show that the 15-kDA tryptic fragment derived from the portion of transformed receptors that do not bind to DNA is itself competent at DNA-binding.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are "misfolded" after removal of hsp90.

After dissociation of cytosolic heteromeric glucocorticoid receptor complexes by steroid, salt, and other methods, only 35-60% of the dissociated receptors can bind to DNA-cellulose. The DNA-binding and non-DNA-binding forms of the dissociated receptors have the same Mr and are phosphorylated to the same extent (Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17347-17349). The basis for the different DNA-binding activities is unknown, but the DNA-binding fraction of the receptor has a more basic pI than the non-DNA-binding fraction (Smith, A. C., Elsasser, M. S., and Harmon, J. M. (1986) J. Biol. Chem. 261, 13285-13292). We have separated the non-DNA-binding state of the receptor from the DNA-binding state and then cleaved it with trypsin and chymotrypsin. We find that the 15-kDa tryptic fragment derived from the non-DNA-binding state of the dissociated receptor is fully competent in binding DNA, whereas the 42-kDa chymotryptic fragment containing both the hormone-binding and DNA-binding domains does not bind DNA. Trypsin cleavage of the molybdate-stabilized untransformed receptor also yields a 15-kDa fragment that is fully competent in binding DNA. Reducing agents do not restore DNA-binding to the non-DNA-binding fraction of the receptor and the hormone-binding domain can be separated from the DNA-binding domain on nonreducing gel electrophoresis. These results argue that the two domains are not linked by disulfide bridges, and they are consistent with the proposal that there are two least energy states of folding after dissociation of hsp90. A significant portion of the receptors is "misfolded" in such a manner that the steroid binding domain is directly preventing DNA-binding activity.     

Relationship between glucocorticoid receptor steroid-binding capacity and association of the Mr 90,000 heat shock protein with the unliganded receptor

Treatment of rat liver cytosol with hydrogen peroxide (H2O2) or sodium molybdate (MoO4(2-)) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25 degrees C. Dithiothreitol (DTT) prevents the stabilization of receptors by H2O2. Heating (25 degrees C) of immune pellets formed by immunoadsorption of L-cell murine glucocorticoid receptor complexes to protein-A-Sepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heat shock protein (hsp90) from the steroid binding protein. Such thermal-induced dissociation of hsp90 is inhibited by H2O2. Pretreatment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H2O2 to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.     

The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated in vivo

The glucocorticoid receptor is phosphorylated, but the precise location of the phosphorylated groups is unknown. We cultured AtT-20 cells in medium containing [32P]-orthophosphate and used immunoaffinity methods to isolate the intact receptor and a tryptic fragment containing the DNA binding domain. Analysis of the intact receptor, co-labeled with the affinity ligand dexamethasone-mesylate, confirmed that the receptor was phosphorylated. Isolation of the DNA binding domain by trypsinization and immunopurification showed that it was not phosphorylated. Interestingly, a non-immunoreactive phosphorylated fragment similar in size to the DNA-binding fragment was observed. Our results suggest that phosphorylation of the DNA binding domain of the glucocorticoid receptor is not essential for hormone action.     

Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties

We have investigated the influence of the N-terminal domain of the 94-kDa glucocorticoid receptor on the DNA:receptor interaction. An alpha-chymotrypsin-induced 39-kDa receptor fragment, containing the hormone and DNA binding domains, binds DNA with a reduced specificity compared to the intact 94-kDa receptor. Various footprinting assays did not reveal any qualitative differences when comparing the DNA contact points made by the two different receptor entities. Like the intact receptor, the 39-kDa receptor fragment binds as a dimer to DNA. Glutaraldehyde cross-linking demonstrated a difference in the protein:protein contacts of the two homodimers. Furthermore, the dimeric 94-kDa receptor did not recognize a half-DNA site, while the dissociated 94-kDa receptor dimer and the dimeric 39-kDa receptor fragment allowed binding to such a site. These results suggest that the loss of the N-terminal domain of the receptor affects the steric arrangement and/or rigidity of the two DNA binding domains of the receptor homodimer, resulting in a decreased DNA binding specificity of the 39-kDa receptor fragment.     

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.     

Phosphorylated sites within the functional domains of the approximately 100-kDa steroid-binding subunit of glucocorticoid receptors

The steroid-binding subunit of the glucocorticoid receptor is known to be a approximately 100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. When isolated from WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel et al., 1987). To identify the domains that contain these phosphorylated sites, we have analyzed the phosphate content of selected proteolytic fragments of the approximately 100-kDa steroid-binding protein from nonactivated and activated receptors. The approximately 100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of [32P]orthophosphate was covalently labeled with [3H]dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin digestion of this protein yields a approximately 45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both 32P and 3H. Trypsin digestion of the protein yields a approximately 29-kDa fragment encompassing the steroid-binding domain but not the DNA-binding domain of the approximately 100-kDa protein, which also contained both 32P and 3H. The 32P/3H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the glucocorticoid receptor DNA-binding domain with DNA as a dimer is mediated by a short segment of five amino acids

We have previously shown that protein-protein interactions mediate cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element (Dahlman-Wright, K., Siltala-Roos, H., Carlstedt-Duke, J., and Gustafsson, J.-A. (1990) J. Biol. Chem. 265, 14030-14035). The cooperativity of DNA binding is lost when the distance between the two half-sites constituting a glucocorticoid responsive element is altered or when their relative orientation is changed. We show here that mutations in the responsive element which interfere with cooperative DNA binding by the glucocorticoid receptor DNA-binding domain in vitro also abolish transactivation by the full length glucocorticoid receptor in vivo. We also identify a short segment in the proximity of one of the bound zinc ions that is required for cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element. We suggest that this segment is involved in dimer formation of the native glucocorticoid receptor and that it is important for correct positioning of the dimeric molecule on the double helix of DNA.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor

Using L cell glucocorticoid receptors that have been immunopurified by adsorption to protein A Sepharose with a monoclonal antireceptor antibody, we have developed an assay to study the requirements for maintenance of steroid-binding capacity. After rapid purification by immunoadsorption, heteromeric receptor complexes retain the ability to bind glucocorticoid hormone. When the receptor complexes are warmed at 20 degrees C, steroid-binding capacity is lost, and the 90-kDa heat shock protein (hsp90) dissociates from the receptor. The rates of both temperature- and salt-dependent dissociation of hsp90 parallel the rates of loss of hormone-binding activity. Molybdate and hydrogen peroxide stabilize the hsp90-receptor complex against temperature-dependent dissociation. Molybdate, however, is much more effective in stabilizing steroid-binding capacity than peroxide. Receptors that have been inactivated in the absence of molybdate or peroxide cannot be reactivated. Inactivation of steroid-binding capacity occurs in the presence or absence of reducing agent, and inactivation is not accompanied by receptor cleavage or dephosphorylation. Under no conditions does an hsp90-free receptor bind steroid. Receptor bound to hsp90 can be cleaved to the 27-kDa meroreceptor in the presence of molybdate with retention of both hsp90 and steroid-binding activity. These observations lead us to propose that hsp90 is necessary but not sufficient for maintaining a competent high affinity glucocorticoid-binding site. Although the 27-kDa meroreceptor fragment is not itself sufficient for a competent binding site, it is sufficient when it is associated with hsp90.     

Immunochemical detection of unique proteolytic fragments of the chick 1,25-dihydroxyvitamin D3 receptor. Distinct 20-kDa DNA-binding and 45-kDa hormone-binding species

We have characterized proteolytic fragments of the chick intestinal 1,25-dihyroxyvitamin D3 (1,25-(OH)2D3) receptor, produced through either exogenous or endogenous protease action, utilizing a variety of physical and functional assays coupled to immunoblot detection methodology. Treatment of intestinal cytosol with increasing concentrations of trypsin resulted in a progressive diminishment of the 60-kDa receptor concomitant with the appearance of a 20-kDa fragment reactive by Western blot analysis to an anti-1,25-(OH)2D3 receptor monoclonal antibody. Cleveland analysis supported the receptor-origin of this 20-kDa fragment: a common immunoreactive species of 12 kDa could be generated by Staphylococcus aureus V8 protease treatment of the intact 60-kDa receptor as well as the 20-kDa proteolytic product. The 20-kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60-kDa receptor and, therefore, may contain the functional DNA-binding domain of the chick 1,25-(OH)2D3 receptor. Thus, this fragment likely represents the complement of a larger hormone-bound fragment that we have previously described (Allegretto, E. A., and Pike, J.W. (1985) J. Biol. Chem. 260, 10139-10145). In contrast to the exogenous effect of trypsin, incubation of cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. Cleveland analysis was consistent with the 60-kDa receptor derivation of the 45-kDa fragment. This species retained the hormone-binding site and the antibody determinant but was devoid of DNA-binding activity. Moreover, it generated neither the trypsin-dependent 20-kDa fragment nor the V8 protease-dependent 12-kDa species and, therefore, was derived from the opposite end of the receptor molecule. These data have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the functional domains for hormone binding and DNA binding.     

Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.     

The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor

Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur.     

Topographical characterization of the domain structure of the bovine adrenal atrial natriuretic factor R1 receptor

We have studied the structure and function of the membrane atrial natriuretic factor R1 (ANF-R1) receptor using limited proteolysis and exoglycosidase treatment. Limited digestion with trypsin of the receptor from bovine adrenal zona glomerulosa membranes resulted in the conversion of the native 130-kDa receptor into a single membrane-associated ANF-binding proteolytic fragment of 70 kDa. The 70-kDa fragment bound ANF with enhanced binding affinity but retained intact ANF-R1 pharmacological specificity and was still sensitive to modulation by amiloride. Trypsin treatment of the membranes produced a dual effect on ANF binding. Low concentrations of trypsin (less than or equal to 25 micrograms/mg of protein) increased ANF binding while higher concentrations dose dependently reduced the binding of the hormone. The increase of ANF-binding activity was associated with the formation of the 70-kDa fragment while the loss of ANF binding paralleled the degradation of the 70-kDa fragment. Low concentrations of trypsin drastically decreased the ANF-sensitive guanylate cyclase activity of the membrane fraction. This loss of catalytic activity strongly correlated with the formation of the 70-kDa tryptic fragment. We also evaluated the effect of ANF binding on the susceptibility of the receptor to proteolytic cleavage. The occupied receptor exhibited a greater sensitivity to trypsin digestion than the unoccupied protein, consistent with the hypothesis that hormone binding induces an important conformational change in the receptor structure. On the other hand, the 70-kDa fragment was much more resistant to proteolysis when occupied by ANF, suggesting that the ANF-binding domain forms a very compact structure.(ABSTRACT TRUNCATED AT 250 WORDS)