No abstract

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)

Studies of hsp90 in yeast have supported the notion that this chaperone plays a critical role in signaling by steroid receptors. One limitation to these studies is that yeast expressing hsp90 mutants may also be deficient in fundamental cellular functions of the chaperone required for steroid-dependent induction of transcription. In this work, we have prepared mutants of the glucocorticoid receptor (GR) that permit analysis of hsp90 binding and transcriptional activity in cells with normal chaperone function. Our previous data supported a model in which hsp90 binds to the receptor steroid binding domain according to a two-site model. By amino acid mutagenesis of these two sites, we have now generated three receptor mutants and analyzed their function. Upon their translation in vitro, all three mutants interacted with hsp90 similarly to the wild-type receptor. However, one mutant, P643A (GRo), was of particular interest because, although it showed normal steroid binding and transformation to a glucocorticoid response element-specific DNA binding form, it was remarkably deficient in nuclear translocation and transcriptional function at 37°C. Furthermore, GRo⅐hsp90 heterocomplexes formed in vivo or assembled under cell-free conditions were much less stable than wildtype GR⅐hsp90 heterocomplexes. Our results demonstrate that Pro-643 plays a critical role in both stabilizing the receptor⅐hsp90 complex and in permitting an efficient nuclear translocation and, thus, support the concept that the chaperone is an integral component of the steroid-receptor signaling pathway.The untransformed form of steroid receptors, such as the glucocorticoid receptor (GR), 1 exists in cells as a large (9 S) heteromeric complex containing the 90-kDa heat-shock protein (hsp90) (1-5), p23, and one of several immunophilins (for review of receptor heterocomplex structure, see Refs. 6 -8). Upon steroid binding, hsp90 dissociates from the original multicomponent structure (3, 9), and the steroid-binding protein is transformed to its nuclear, DNA binding, and transcriptionally active form (9, 10). Given the correlation between hsp90 association and inactivity of the receptor, an inhibitory role for this association has been typically postulated (1, 2, 11). In apparent contradiction with the inhibitory model of hsp90, genetic analysis in yeast expressing low levels of hsp90 (12) or hsp90 point mutants (13,14) indicates that hsp90 is required for steroid receptor signaling in vivo. Moreover, the drug geldanamycin, a benzoquinone ansamycin, specifically binds to hsp90 and, by interference with critical steps in the hsp90-assisted folding, causes significant functional defects in the substrate proteins for hsp90. In the case of GR, geldanamycin suppresses the steroid binding (15, 16), nuclear translocation (16), and transcriptional functions (15, 17) of the receptor. Also, the results of experiments where both the GR and the mineralocorticoid receptor are expressed in bacteria (18, 19) support a permissive role of hsp90. Although affinity-purified ...

Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.

Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and Gu, ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 3046 in chol B and residues 102-I 18 in human-MBP, hMBP, p < 0.0007) and an 11 residue span (residues 3141 in chol B and sequence 29-39 in hMBP, p