DNA-binding State Research Articles

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.

Stabilization in vitro of the untransformed glucocorticoid receptor complex of S49 lymphocytes by the immunophilin ligand FK506

Untransformed steroid receptors are large heteromeric complexes which have been shown to contain the mammalian heat shock proteins hsp56, hsp70 and hsp90. Based on functional and sequence homology studies, it was recently discovered that hsp56 also belongs to the FKBP class of immunophilin proteins, which are thought to mediate the actions of the immunosuppressive drugs FK506 and rapamycin. This discovery has led to the speculation that FK506 and related drugs could influence the actions of steroid receptors. In this work, we have examined the effects of FK506 on the transformation and hormone-binding properties of glucocorticoid receptors (GR) present in the cytosolic fraction of mouse S49 lymphocyte cells. Based on immunoprecipitation studies, it was found that hsp56 was indeed a component of untransformed GR complexes in S49 cytosols. It was also found that the untransformed but not the transformed GR was retained following affinity chromatography with FK506-affigel resin, reinforcing the possibility that hsp56 within the untransformed GR complex could be a target for the actions of FK506. Using a DNA-cellulose-binding assay, FK506 exhibited a 60% inhibition of dexamethasone (Dex)-induced transformation of the GR to the DNA-binding state, while sodium molybdate, a transition metal oxyanion known to stabilize GR complexes, was 100% effective. This inhibition of GR transformation by FK506 was shown to correlate with an inhibition of Dex-induced GR/hsp90 dissociation, with 10 microM FK506 preventing 48% of the GR/hsp90 complexes from dissociating. Scatchard analysis of GR hormone-binding function was performed, with FK506 treatment of cytosols causing Kd values to decrease (3.36 nM) as compared to vehicle (8.42 nM) and no-addition (9.82 nM) controls. Taken together, our results suggest that FK506 can stabilize the untransformed GR complex of S49 cells and that this stabilization in turn results in an increase in GR ligand-binding affinity. Although we speculate that these actions of FK506 on the GR complex are mediated by the associated hsp56 component, other possible mechanisms are also discussed.

Effects of neurohormonal stress and aging on the activation of mammalian heat shock factor 1.

The mammalian heat shock response has been investigated extensively using tissue culture cells with only a limited amount of information available on animals and intact tissues. The neurohormonal stress response mediated by the hypothalamic-pituitary-adrenal axis leads to the activation of heat shock factor (HSF) in rat adrenal tissue. Here we show through the use of antibodies specific to each member of the HSF family that restraint-induced stress in intact Wistar rats and adrenocorticotropic hormone treatment of hypophysectomized animals leads to the activation of HSF1 monomers to trimers with DNA-binding activity. Because HSF1 is also the target factor for metabolic and environmental stress, these data reveal an intersection of pathways leading to HSF1 activation. Comparison of the biochemical properties and levels of HSF1 in the Wistar and Fischer 344 rat strains reveals that HSF1 is constitutively present in an activated DNA-binding state in the adrenals of Fischer 344 rats. During aging, the levels of HSF1 remain constant, yet the transcription factor from aged animals exhibits a decreased ability to bind DNA.

Expression of heat shock factor 2 in mouse testis: potential role as a regulator of heat-shock protein gene expression during spermatogenesis.

We have examined the expression and function of heat shock transcription factor 2 (HSF2) in spermatogenic cells of mouse testis. The results of in situ RNA hybridization analysis, RNA filter hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicate that HSF2 mRNA expression in testis is subject to developmental and cell type-dependent, as well as stage-dependent, regulation. Localized expression of HSF2 mRNA in testis first appears between Day 14 and Day 21 of postnatal development. In adult testis, HSF2 mRNA is found at highest levels in spermatocytes and round spermatids. Immunocytochemical staining and gel mobility shift analysis demonstrate that HSF2 protein is localized to the nuclei of spermatocytes and round spermatids and that this transcription factor exists in testis in a constitutively active DNA-binding state. We further demonstrate that the constitutive HSF2 DNA-binding activity present in testis is able to interact with promoter sequences of the hsp70.2 gene, a testis-specific member of the hsp70 gene family. Taken together, our results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during spermatogenesis.

3,3',4,4'-Tetrachlorobiphenyl inhibits proliferation of immature thymocytes in fetal thymus organ culture.

The environmental pollutant 3,3',4,4'-tetrachlorobiphenyl (TCB) leads to thymic atrophy and immunosuppression, the former possibly causing the latter. TCB binds to the cytosolic aryl-hydrocarbon receptor (AhR) and transforms it into a DNA-binding state. The development of fetal thymocytes is severely affected by TCB and other AhR-binding xenobiotics, leading to a skewed pattern of thymocyte maturation stages. Murine thymocyte proliferation after exposure to TCB was studied in fetal thymus organ culture (FTOC). C57BL/6 fetus thymic lobes from day 15 of gestation were explanted and grown for 2, 4, 6, and 8 days in organ culture in the presence or absence of 3.3 microM TCB. Subsets of thymocytes were defined by CD4 and CD8 surface markers, and their cell cycle was analysed by DNA staining with 7-amino-actinomycin D (7-AAD). Exposure of fetal thymi in vitro to 3.3 microM TCB significantly reduced the total number of thymocytes, and fewer thymocytes were in S/G2M phase. The inhibition of cell proliferation induced by TCB treatment affected mainly the CD4-CD8- (double-negative, DN) and CD4-CD8+ (single-positive, SP) subsets, and these inhibition appeared mainly in more immature thymocytes, i.e. DNCD3- and CD8+CD3- subpopulations, whereas no effect of TCB on CD4+CD8+ (double-positive, DP) cell proliferative activity was observed. Analysis of the relation of cell proliferation and development of subsets in differentiating fetal thymocytes suggests that TCB enhanced thymocyte differentiation into mature CD8+ cells.

Mutant cell lines unresponsive to alpha/beta and gamma interferon are defective in tyrosine phosphorylation of ISGF-3 alpha components.

The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-alpha and -gamma. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-alpha/beta and IFN-gamma systems.

Mutant Cell Lines Unresponsive to Alpha/Beta and Gamma Interferon Are Defective in Tyrosine Phosphorylation of ISGF-3α Components

The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-alpha and -gamma. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-alpha/beta and IFN-gamma systems.

Real‐time DNA binding measurements of the ETSl recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.

Proof that hsp70 is required for assembly of the glucocorticoid receptor into a heterocomplex with hsp90.

Incubation of immunopurified glucocorticoid receptor with rabbit reticulocyte lysate forms a complex between the receptor and hsp90, with simultaneous conversion of the receptor from a non-steroid binding but DNA binding state typical of the transformed receptor back to the steroid binding, non-DNA binding state typical of the untransformed receptor (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). The receptor heterocomplex formed by the lysate also contains hsp70 and is formed in an ATP-dependent and cation-selective manner (Hutchison, K. A., Czar, M.J., Scherrer, L. C., and Pratt, W.B. (1992) J. Biol. Chem. 267, 14047-14053). In this work, we selectively depleted reticulocyte lysate of hsp70 by passing it through a column of ATP-agarose. The hsp70-depleted lysate contains hsp90, but it cannot form a receptor-hsp90 heterocomplex. hsp70 purified from mouse L cells binds to immunopurified glucocorticoid receptor but does not convert it to the steroid binding state. Addition of purified hsp70 to the hsp70-depleted lysate reactivates the heterocomplex assembly system, permitting formation of a receptor-hsp90-hsp70 complex, with the receptor being returned to the high affinity steroid-binding conformation. These data are consistent with a model in which the protein-unfolding activity of hsp70 is required for hsp90 binding to the hormone binding domain of the glucocorticoid receptor. The hsp56 immunophilin component of the native receptor heterocomplex is also present in the reconstituted receptor heterocomplex in an hsp70-dependent manner. In addition to hsp70, other as yet unidentified factors in reticulocyte lysate are required for receptor heterocomplex assembly.

Identification of the C-terminal activator domain in yeast heat shock factor: independent control of transient and sustained transcriptional activity.

In yeast, heat shock factor (HSF) is a trimer that binds DNA constitutively but only supports high levels of transcription upon heat shock. The C-terminal regions of HSF from Saccharomyces cerevisiae and Kluyveromyces lactis are unconserved yet both contain strong transactivators which are correctly regulated when substituted for each other. We have performed high resolution mapping of these activator domains which shows that in K.lactis HSF (KlHSF) activity can be located to a confined short domain, while in S.cerevisiae HSF (ScHSF) two separate regions are required for full activity. Alignment of the activator domains reveals similarity, as both overlap potential leucine zipper motifs (zipper C) with a distribution of hydrophobic residues similar to two highly conserved N-terminal domains which mediate HSF trimerization (zippers A and B). In higher eukaryotes a C-terminal leucine zipper is required to maintain HSF in a monomeric and non DNA-binding state under normal conditions and we therefore address the regulatory roles of the three leucine zipper motifs in KlHSF. Whilst the longest and most N-terminal of the trimer region zippers, A, is dispensable for regulation, mutation of a single leucine in zipper B makes HSF constitutively active. In contrast to the situation in higher eukaryotes disruption of zipper C has no observable regulatory effect and therefore, although an intramolecular contact between zippers B and C cannot be ruled out, such contact is not required for restraining the C-terminal activator domain. We furthermore find that deletions which abolish activator potential of the C-terminus render the host strain temperature sensitive. However, deletion of a double proline-glycine motif in the activator, whilst leaving HSF unable to respond to heat shock, does not cause temperature sensitivity. This result demonstrates that independent mechanisms control the transient and sustained activities of HSF.

Negative Co-dominant Inhibition of recA Protein Function: Biochemical Properties of the recA1, recA13 and recA56 Proteins and the Effect of recA56 Protein on the Activities of the Wild-type recA Protein Function in Vitro

We have investigated the biochemical properties of several Escherichia coli mutant recA proteins that display a null phenotype. These are the recA1, recA13 and recA56 proteins, each of which carries a single missense mutation. These proteins all share a common defect which is the inability to adopt the high affinity DNA binding state normally elicited by the nucleotide cofactor ATP. Consequently, other than the ability to bind ssDNA, they possess none of the in vitro enzymatic activities of recA protein. However, each protein has characteristics that are unique, leading to the conclusion that the observed mutant phenotypes arise through fundamentally different mechanisms. Despite the magnitude of these defects, the recA56 protein is able to differentially inhibit various activities of wild-type recA protein. Incorporation of recA56 protein into a presynaptic filament with the wild-type recA protein does not affect the ability of the wild-type protein to hydrolyze ATP, as judged by the turnover number ( k cat), provided that the ssDNA concentration is not limiting; however, the affinity of wild-type recA protein for ATP is lowered by the presence of recA56 protein. Similarly, the ability to cleave lexA protein is only modestly inhibited. However, both the ability to compete with SSB protein for ssDNA binding sites and the DNA strand exchange activity of wild-type recA protein are severely inhibited by the presence of recA56 protein. These results suggest that individual monomeric components of the recA protein-DNA filament are translated through protein-protein contacts to become macroscopic properties of the filament.

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of the aryl hydrocarbon receptor to a DNA-binding form is accompanied by release of the 90 kDa heat-shock protein and increased affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor (AhR) elicits a sequence of poorly defined molecular events that ultimately yield a heteromeric transformed AhR that is active as a transcription factor. We have previously developed a model of the ligand-initiated transformation of the AhR to the DNA-binding state based on characterization of several forms of the AhR with respect to their physicochemical properties and DNA-binding affinities. The present studies were designed to determine whether, and at what stage, this process of transformation alters the receptor's affinity for TCDD. In rat hepatic cytosol, approx. 10% of the TCDD specifically bound to the AhR rapidly dissociated (t1/2 approximately 1 h), while the remainder was only slowly dissociable (t1/2 approximately 70 h). The isolated DNA-binding forms of the receptor (monomeric and transformed) bound TCDD very tightly (t1/2 > 100 h), whereas TCDD was dissociable from the non-DNA-binding receptor form(s). A lower incubation temperature (0-4 degrees C) and the presence of molybdate partially stabilized the non-DNA-binding fraction of the TCDD.receptor complex and also enhanced TCDD dissociation in crude cytosol. Immunoprecipitation of the different AhR forms with an anti-AhR antibody and immunoblotting with antibody to the 90 kDa heat-shock protein (hsp90) demonstrated that hsp90 was associated with the unoccupied receptor complex as well as with a fraction of the non-DNA-binding TCDD.receptor complex; isolated DNA-binding forms did not contain detectable hsp90. We conclude that while hsp90 remains associated with the AhR, TCDD is readily dissociable; following release of hsp90, however, TCDD becomes very tightly bound, and remains so upon completion of transformation.

Functions of estrogens and anti‐estrogens in the rat endometrial adenocarcinoma cell lines RUCA‐I and RUCA‐II

Inbred rats of the DA/Han and BDII/Han strains have been proposed as suitable model systems for studying hormonal carcinogenesis, because they die mainly from hormone-dependent endometrial adenocarcinoma. Here we characterize the RUCA-I cell line derived from an endometrial adenocarcinoma of an inbred DA/Han rat and the RUCA-II cell line derived from an endometrial adenocarcinoma of an inbred BDII/Han rat. The RUCA-I cell line, if transplanted to the neck of female DA/Han rats, gives rise to endometrial adenocarcinomas at the ectopic site. The morphology of these ectopically grown tumors is predominantly of the moderately differentiated sub-class. In contrast, ectopic tumor growth of the RUCA-II cell line can be observed only if cells are transplanted to athymic nude mice. Biochemically, both cell lines are characterized by the stable expression of estrogen receptors. However, no statistically significant mitotic response of RUCA-I and RUCA-II cells to estradiol was measurable, and no induction of expression of the progesterone receptor by estradiol was detectable, although estradiol transformed the estrogen receptor into its stable DNA-binding state. In contrast, the rate of proliferation of RUCA-I but not of RUCA-II cells was reduced in the presence of 10(-6) M tamoxifen. From these results we conclude that (i) both cell lines, RUCA-I and RUCA-II, represent a new and promising endometrial tumor model; (ii) the mechanism of the hormone-dependent growth regulation of RUCA-I and RUCA-II cells is obviously impaired; (iii) the RUCA-I cell line appears to be a suitable model system for the study of molecular aspects of estrogen- and tamoxifen-dependent gene expression.

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.

Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins

The recA protein of E coli participates in several diverse biological processes and promotes a variety of complex in vitro reactions. A careful comparison of the phenotypic behavior of E coli recA mutations to the biochemical properties of the corresponding mutant proteins reveals a close parallel both between recombination phenotype and DNA strand exchange and renaturation activities, and between inducible phenomena and repressor cleavage activity. The biochemical alterations manifest by the mutant recA proteins are reflected in the strength of their interaction with ssDNA. The defective mutant recA proteins fail to properly assume the high-affinity DNA-binding state that is characteristic of the wild-type protein and, consequently, form less stable complexes with DNA. The mutant proteins displaying an ‘enhanced’ activity bind ssDNA with approximately the same affinity as the wild-type protein but, due to altered protein-protein interactions, they associate more rapidly with ssDNA. These changes proportionately affect the ability of recA protein to compete with SSB protein, to interact with dsDNA, and, perhaps, to bind repressor proteins. In turn, the DNA strand exchange, DNA renaturation, and repressor cleavage activities mirror these modifications.

Subunit structure of the glucocorticoid receptor and activation to the DNA-binding state

Glucocorticoid receptors of S49.1 mouse lymphoma cells were analyzed under a variety of conditions. The complexes with an agonist or a steroidal antagonist can be formed in cytosolic extracts, they are of high molecular weight, M r ∼ 330,000 and have a Stokes radius of 82 Å. Cross-linking by several agents stabilized this structure against subunit dissociation which produces the activated receptor form of 60 Å and DNA-binding ability. Careful analysis of intermediate cross-linked forms lead to the conclusion that the large receptor structure is a hetero-tetramer consisting of one hormone-bearing polypeptide of M r ∼ 94,000, two 90 kDa subunits and a protein component of M r ∼ 50,000. The 90 kDa subunits are the heat shock protein hsp90. The high molecular weight receptor form also exists in intact cells are revealed again by cross-linking. The cytosolic complex with the antagonist can become activated to the DNA-binding form upon warming but simultaneously looses the ligand. Ligand rebinding does not occur subsequent to receptor dissociation. Upon incubation of intact cells at 37°C with agonist or antagonist the respective receptor-ligand complexes are formed. The agonist complex is immediately activated, however, the antagonist complex remains stable in the undissociated state. This explains the biological effect of the antagonist.

Transient elevation of estrogen receptors in the neonatal rat hippocampus

The presence of sex differences in hippocampal morphology and function suggests that this brain region may be sensitive to the organizational actions of gonadal steroids. We therefore examined the postnatal development of estrogen receptor (ER) in the rat hippocampal formation. ER was measured by the in vitro binding of [ 3H]estradiol to a cytosolic preparation. Radioinert R2858 (moxestrol) was used to determine nonspecific binding. Hippocampal ER concentrations increased from birth through postnatal day (PND) 4 when levels peaked (10.05 ± 1.2 fmol/mg protein); these were maintained through PND-7 (9.45 ± 1.4) and declined thereafter to low levels characteristic of the adult (2.05 ± 0.35). This ontogenic profile is similar to that found is several neocortical regions, as well as in the cingulate cortex, but is distinct from that observed in the hypothalamus, where ER levels remain high in the adult. Saturation analysis of PND-7 hippocampal cytosols demonstrated a single, high affinity binding site ( K d: 5.51 ± 1.7 × 10 −10 M). [ 3H]Estradiol binding was specific in that it was displaced by radioinert R2858, diethylstilbestrol (DES), and 17β-estradiol but not by nonestrogenic steroids. Significantly greater ER levels were found in hippocampal nuclear extracts from DES-treated PND-7 animals compared to controls (9.74 ± 2.27 vs. 0.49 ± 0.24 fmol/mg DNA, P < 0.01). The presence of functional ER was also shown by the ability of receptors to be retained on DNA cellulose. DNA cellulose column chromatography elution profiles for PND-7 hippocampal and medial basal hypothalamic (MBH) cytosols following incubation with [ 3H]estradiol were similar. The presence of elevated hippocampal ER levels during the perinatal critical period and evidence of functional transformation to the DNA binding state following DES treatment in vivo or estrogen incubation in vitro suggests that the hippocampus is a potential substrate for estrogen-mediated organizational events.

Gene Regulation by Receptors Binding Lipid-Soluble Substances

The model for estrogen action proposed by Jensen & Gorski more than twenty years ago has served as a unifying theory to explain regulation of gene expression by many steroid hormones (34, 48). Each hormone triggers an allosteric change in a specific receptor protein converting it from a low affinity DNA-binding state to one with relatively higher affinity for DNA elements in the vicinities of target genes. This tighter DNA binding interac­ tion is thought to catalyze the assembly of transcriptional complexes at nearby sites and activate target cell gene expression (111). Despite extensive characterization of the receptor proteins and their target DNA sequences, our knowledge of how the activated receptor initiates transcription from hormone­ responsive genes is still incomplete (for other reviews see 5, 14, 24). Recent identification of several steroid hormone receptor genes has helped to uncover a large family of nuclear receptor or ligand-dependent transcription

Glucocorticoid receptor activation during exercise in muscle

This investigation was undertaken to evaluate whether endurance running of the type known to retard the muscle atrophy associated with glucocorticoid excess inhibits activation of glucocorticoid-receptor complexes to a DNA binding state. Female adrenalectomized rats received an injection (50 microCi/100 g body wt ip) of [3H]triamcinolone acetonide and remained sedentary or were immediately exercised by endurance running at 23 m/min for up to 90 min. Receptor activation, as quantified by binding to DNA-cellulose, steadily increased from 10-20% of the receptors capable of binding DNA in uninjected controls to 25-45% by 5 min and to 53-80% by 90 min after receiving the hormone in all muscles studied (fast-twitch red vastus lateralis, fast-twitch white vastus lateralis, slow-twitch soleus, mixed gastrocnemius, and heart). Exercise did not influence the time-course changes in percent activation. When activation was determined from changes in the conformational state of the receptor as measured by diethylaminoethyl-cellulose anion exchange chromatography, there was a similar time-dependent formation of activated receptor forms in all muscle types. However, exercise did not inhibit or delay the appearance of the activated receptor from the unactivated state. These results indicate that glucocorticoid receptor activation occurs at a rate that is independent of both fiber type and delivery of steroid to working muscles during exercise. If exercise alters receptor activation, a longer time period, beyond 90 min of running, or even additional training may be needed for inhibition to be expressed.