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

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

Unspecific DNA binding of the DNA binding domain of the glucocorticoid receptor studied with flow linear dichroism

The unspecific interaction between the DNA-binding domain of the human glucocorticoid receptor and DNA was studied using linear dichroism (LD) and circular dichroism (CD) spectroscopy. The amplitude of the LD signal was found to increase upon addition of protein at ionic strengths less than 60 nM Na+, indicating an increased persistence length of the complex compared to uncomplexed DNA. Analysis of the LD spectrum suggests that the binding does not involve intercalation of tyrosine residues. Evidence of saturation is found at a binding stoichiometry of approximately 5 DNA base pairs per protein monomer.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.     

Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor

The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexpressed and purified from Escherichia coli previously. This fragment has been shown to contain 2.3 +/- 0.2 mol of Zn(II) per mole of protein [Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., & Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR(440-525)] with 113Cd(II) substituted for Zn(II) identifies 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus. The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands. Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear. Addition of EDTA to the protein restores the original two signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping the HSP90 binding region of the glucocorticoid receptor

In animal cells, unliganded steroid receptors are complexed with a 90-kDa heat shock protein, HSP90; hormone binding by the receptor leads to the release of HSP90. We found that the 795-amino acid rat glucocorticoid receptor protein formed oligomeric complexes in vitro upon synthesis in rabbit reticulocyte lysates; these oligomers also dissociated in the presence of hormone. Similar complexes formed when X795, a receptor derivative containing only the C-terminal half (amino acids 407-795) of the protein, was translated in vitro. Moreover, X795 was co-immunoadsorbed from the reticulocyte lysates together with HSP90 by three different anti-HSP90 monoclonal antibodies, indicating that the in vitro translated receptor binds HSP90 and that the interaction occurs within the C-terminal half of the receptor. To localize the HSP90 binding region in greater detail, various deletion mutants of X795 were translated in vitro and assayed for oligomer formation and for co-immunoadsorption with HSP90. The results indicated that HSP90 interacted with the receptor within a subregion of the hormone binding domain, between amino acids 568 and 616. These findings are consistent with the proposal that HSP90 may participate in the mechanism of signal transduction by steroid receptors.     

Mechanism of kappa B DNA binding by Rel/NF-kappa B dimers

The DNA binding of three different NF-kappaB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-kappaB p50/p65 heterodimer is shown here to bind the kappaB DNA target site of the immunoglobulin kappa enhancer (Ig-kappaB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-kappaB complex. The heterodimer binds to the Ig-kappaB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-kappaB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 degrees C and 42 degrees C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-kappaB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.     

Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain.

In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the receptor protein does not influence its DNA-binding affinity, while the C-terminal steroid-binding domain increases the DNA-binding affinity of the receptor molecule. It has previously been shown that both the intact glucocorticoid receptor and the glucocorticoid receptor DNA-binding domain bind to a palindromic glucocorticoid response element on DNA as dimers. It is likely that differences in DNA-binding affinity observed result from protein-protein interactions outside the DNA-binding domain between receptor monomers, as has been shown for the estrogen receptor. We have previously identified a segment involved in protein-protein interactions between DNA-binding domains of glucocorticoid receptors. This, in combination with results presented in this study, suggests that there are at least two sites of contact between receptor monomers bound to DNA. We suggest that the interaction between the DNA-binding domains may act primarily to restrict DNA binding to binding sites with appropriate half-site spacing and that additional stability of the receptor dimer is provided by the interactions between the steroid-binding domains.     

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.     

Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-kappa B ligand

Dermatan sulfate (DS) is a major component of extracellular matrices in mammalian tissues. In the present study, DS demonstrated a high level of binding activity to receptor activator of NF-kappaB ligand (RANKL) and obstructed the binding of RANK to RANKL, determined using a quartz-crystal microbalance (QCM) technique. Further, when mouse bone marrow cells were cultured with RANKL and macrophage colony-stimulating factor, DS suppressed tartrate-resistant acid phosphatase-positive multinucleated cell formation in a dose-dependent manner. In addition, immunoblot analyses revealed that DS reduced the levels of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase protein in mouse osteoclast progenitor cells stimulated with RANKL. Together, these results indicate that DS regulates osteoclast formation through binding to RANKL and inhibition of signal transduction in osteoclast progenitor cells, suggesting that it has an important role in bone metabolism in pathological conditions.     

Peroxisome proliferator-activated receptor gamma-mediated NF-kappa B activation and apoptosis in pre-B cells

The role of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte physiology has been exploited for the treatment of diabetes. The expression of PPARgamma in lymphoid organs and its modulation of macrophage inflammatory responses, T cell proliferation and cytokine production, and B cell proliferation also implicate it in immune regulation. Despite significant human exposure to PPARgamma agonists, little is known about the consequences of PPARgamma activation in the developing immune system. Here, well-characterized models of B lymphopoiesis were used to investigate the effects of PPARgamma ligands on nontransformed pro/pre-B (BU-11) and transformed immature B (WEHI-231) cell development. Treatment of BU-11, WEHI-231, or primary bone marrow B cells with PPARgamma agonists (ciglitazone and GW347845X) resulted in rapid apoptosis. A role for PPARgamma and its dimerization partner, retinoid X receptor (RXR)alpha, in death signaling was supported by 1) the expression of RXRalpha mRNA and cytosolic PPARgamma protein, 2) agonist-induced binding of PPARgamma to a PPRE, and 3) synergistic increases in apoptosis following cotreatment with PPARgamma agonists and 9-cis-retinoic acid, an RXRalpha agonist. PPARgamma agonists activated NF-kappaB (p50, Rel A, c-Rel) binding to the upstream kappaB regulatory element site of c-myc. Only doses of agonists that induced apoptosis stimulated NF-kappaB-DNA binding. Cotreatment with 9-cis-retinoic acid and PPARgamma agonists decreased the dose required to activate NF-kappaB. These data suggest that activation of PPARgamma-RXR initiates a potent apoptotic signaling cascade in B cells, potentially through NF-kappaB activation. These results have implications for the nominal role of the PPARgamma in B cell development and for the use of PPARgamma agonists as immunomodulatory therapeutics.     

Modulation of DNA binding of glucocorticoid receptor by aurintricarboxylic acid

Effects of aurintricarboxylic acid (ATA) were examined on the DNA binding properties of rat liver glucocorticoid-receptor complex. The DNA-cellulose binding capacity of the glucocorticoid-receptor complex was completely abolished by a pretreatment of receptor preparation with 0.1-0.5 mM ATA at 4 degrees C. The half-maximal inhibition (i.d.50) in the DNA binding of [3H]triamcinolone acetonide-receptor complex [( 3H]TARc) was observed at 130- and 40 microM ATA depending upon whether the inhibitor was added prior to or following the receptor activation. The entire DNA-cellulose bound [3H]TARc could be extracted in a concentration-dependent manner by incubation with 2-100 microns ATA. The [3H]TARc remained intact under the above conditions, the receptor in both control and ATA-treated preparations sedimented in the same region in salt-containing 5-20% sucrose gradients. The action of ATA appeared to be on the receptor and not on DNA-cellulose. The DNA-binding capacity of ATA-treated receptor preparations could be recovered upon exhaustive dialysis. The treatment with ATA did not appear to change the ionic behavior of heat activated GRc; the receptor in both control and the ATA-treated preparations showed similar elution profiles. Therefore, ATA appears to alter the binding to and dissociation of glucocorticoid-receptor complex from DNA. The use of ATA should offer a good chemical probe for analysis of the DNA binding domain(s) of the glucocorticoid receptor.