Depletion of rat liver glucocorticoid receptor hormone-binding and its mRNA in sepsis

Glucocorticoid receptor (GR) hormone-binding activity, its physical characteristics, and GR mRNA levels were studied in the liver, brain and muscle of normal (saline-injected) and hypermetabolic septic rats 24 h after the subcutaneous injections of E. coli. The GR levels (hormone-binding activity) declined by about 40%, 56%, and 40% in septic liver, brain, and muscle cytosol, respectively. The mechanism of the decrease in the GR levels in sepsis was studied in liver. The GR levels remained low (45% of control hormone-binding) even after 48 h of E. coli administration. The decrease in the liver GR occurred in the 9S untransformed GR. The 9S GR from septic liver transformed to the 4S form in proportions comparable to the control liver GR. In addition, the 4S GR from control and septic liver was capable of binding to DNA-cellulose to a similar extent. The GR mRNA level in septic liver declined by about 30%. Thus, a decrease in GR hormone-binding activity in sepsis appears to be due to a decline in the steady-state GR mRNA level and not from a change in the qualitative properties of the GR protein.     

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.     

Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain

Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced "super" receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity.     

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.     

Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition

In this work, we used two approaches to localize the 90-kDa heat shock protein (hsp90)-binding site within the hormone-binding domain of the glucocorticoid receptor. In the first approach, derivatives of the glucocorticoid receptor deleted for increasing portions of the COOH terminus were translated in rabbit reticulocyte lysate, and the [35S]methionine-labeled translation products were immunoadsorbed with the 8D3 monoclonal antibody against hsp90. The data suggest that a segment from amino acids 604 to 659 (mouse) of the receptor is required for hsp90 binding. We have recently shown that the internal deletion mutant of the mouse receptor (delta 574-632) binds hsp90, although the complex is somewhat unstable (Housley, P. R., Sanchez, E. R., Danielsen, M., Ringold, G. M., and Pratt, W. B. (1990) J. Biol. Chem. 265, 12778-12781). The two observations indicate that amino acids 574-659 are involved in forming a stable receptor-hsp90 complex and that region 632-659 is especially important. To test this hypothesis directly, we synthesized three peptides corresponding to segments in region 624-665 and three peptides spanning the highly conserved sequence at amino acids 582-617, and we then tested the ability of the peptides to compete for the association of hsp90 with the L cell glucocorticoid receptor. In this assay, the immunopurified hsp90-free mouse receptor is incubated with rabbit reticulocyte lysate, which directs the association of rabbit hsp90 with the mouse receptor, simultaneously converting the receptor to the steroid binding state. All three peptides spanning region 624-665 and a peptide corresponding to segment 587-606 inhibited both hsp90 association with the receptor and reconstitution of steroid binding capacity. The data from all of the approaches support a two-site model for the hsp90-binding site in which the critical contact site occurs in region 632-659, which contains a short proline-containing hydrophobic segment and adjacent dipole-plus-cysteine motif that are conserved among all of the hsp90-binding receptors in the superfamily. A second hsp90 contact site is predicted in region 574-632, which contains the only highly conserved amino acid sequence in the receptor superfamily outside of the DNA-binding domain.     

Mutation detection of the human glucocorticoid receptor alpha gene area coding for the hormone-binding domain by denaturing gradient gel electrophoresis

Mutations in the hormone-binding domain of the human glucocorticoid receptor alpha (hGRalpha) gene have been detected in a variety of glucocorticoid resistance syndromes. Using the denaturing gradient gel electrophoresis technique, we developed a sensitive method for the detection of alterations in the gene area coding for the whole hormone-binding domain and part of the DNA-binding domain of the hGRalpha. This method can be applied for screening of glucocorticoid receptor gene alterations in glucocorticoid-dependent diseases.     

Expression of the mouse glucocorticoid receptor and its role during development

Genes encoding enzymes involved in gluconeogenesis are activated in liver shortly after birth by the synergistic effect of glucagon and glucocorticoids. This induction is achieved by the synergistic action of hormone responsive and liver-specific enhancer elements. In the case of glucocorticoids, this enhancer is composed of a glucocorticoid-response element (GRE) and a number of cell-specific hepatocyte nuclear factor 3 (HNF-3) binding sites. The GRE binds the ligand-activated glucocorticoid receptor (GR) which is ubiquitously expressed and the HNF-3 element binds a cell-specific protein factor. To further understand the role of cell-specific glucocorticoid signalling in the perinatal period and earlier during development we have studied the expression of the mouse GR gene. The gene has been cloned and fully characterized. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected back to day 9.5 of mouse development. The role of GR during mouse development has been further analysed by disruption of the GR gene in vivo by homologous recombination in mouse embryonic stem cells.     

Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.     

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.     

Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association.

The transforming protein of the avian sarcoma virus UR2 is a 68-kDa transmembrane tyrosine protein kinase. We examined the relationship between membrane localization and transforming activity of P68 by changing Val-168-Val-169 in its hydrophobic domain into Asp-168-Glu-169. The resulting transmembrane (TM) mutant (P68TM) lost transforming activity toward chicken embryo fibroblasts (CEF). We found that the mutant protein was expressed and rapidly degraded into a smaller form which was still membrane associated and kinase active. The instability of the TM mutant protein is a phenomenon only manifested in CEF, because the same mutant protein was expressed with efficiency and stability similar to those of the wild-type protein in a transient expression system in COS cells. However, there are several differences between the wild-type and the TM mutant proteins in COS cells. The wild-type protein is more heavily phosphorylated and associated with membrane fractions in a cotranslational manner. It is enzymatically active when recovered from membrane fractions. The TM mutant protein is less phosphorylated, more labile toward protease degradation, and delayed in membrane association, with a lag period of 30 min or longer, and has little kinase activity when recovered from membrane fractions. Most of the kinase-active TM mutant protein was found in the cytosol fractions. Despite the delay, most of the TM protein in COS cells was found to be membrane associated, and its orientation on the cell surface was similar to that of the wild-type protein. It is probable that loss of the CEF-transforming activity of the TM mutant protein is due to its susceptibility to protease degradation resulting from improper membrane association of the newly synthesized product. The differences in the kinetics of membrane association and the distribution of kinase activity in COS cells might not be directly applicable in explaining the inability of the TM mutant to transform CEF but are intriguing as regards protein biosynthesis and translocation. The difference between CEF and COS cells implies that different factors or pathways are involved in the biosynthesis and processing of the TM mutant protein in these two cellular environments. Changes of P68TM in the kinetics of membrane association indicate that the transmembrane domain of ros, besides functioning as a membrane anchor, also plays a role in directing initial membrane association.     

Single point mutations of the sodium channel drastically reduce the pore permeability without preventing its gating

1. Two mutants of the sodium channel II have been expressed inXenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine.2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the openchannel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions.3. Mutant D384N has a very low permeability for any of the following ions: Cl^–, Na⁺, K⁺, Li⁺, Rb⁺, Ca²⁺, Mg²⁺, NH₄ ⁺ , TMA⁺, TEA⁺. However, asymmetric charge movements similar to the gating currents of the Na⁺-selective wild-type are still observed.4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.Abbreviations TTX tetrodotoxin; Na⁺, sodium; K⁺, potassium; - NFR normal frog Ringer - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis(-amino-ethyl ether) N,N,N',N'-tetra acetic acid - TEA tetraethylammonium - TMA tetramethylammonium;I _g , gating current; , single-channel conductance