Identification of a protein from rat liver cytosol that enhances activation of the glucocorticoid receptor.

We have identified a factor from rat liver cytosol that enhances the DNA-cellulose-binding ability of the glucocorticoid receptor and lowers the sedimentation value from 9-10 S to 4-5 S. Cytosol is prepared in the presence of molybdate, and unactivated receptor is isolated by chromatography on DEAE-cellulose in the presence of molybdate. This receptor sediments at 9-10 S and has little affinity for DNA. If the molybdate is removed and the receptor is incubated at 25 degrees C with the low-salt wash of the DEAE-cellulose column, DNA binding is enhanced by 50-600% relative to controls incubated with buffer only. In addition, the factor present in the low-salt wash converts the 9-10 S receptor into a mixture of 5 S and 4 S forms. The factor must be present during the incubation in order to exert its maximal effect. Factor added after the incubation has only marginal effects on the DNA-binding ability of the receptor, indicating that the factor does not increase the DNA-binding ability of activated receptor. Moreover, the factor is significantly less effective on receptor that has been activated before incubation with the factor. These results suggest that the factor acts as an activation enhancer. Preliminary characterization indicates that the activation enhancer is a trypsin-sensitive protein of approx. 70,000 Da, whose activation-enhancing properties are inhibited by ATP. RNAase A, which has effects similar to those described above on the 7-8 S receptor, does not mimic the effects of the activation enhancer on the 9-10 S receptor.

Isolation of ribonucleic acid from the unactivated rat liver glucocorticoid receptor.

We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.

The ability to convert the 4 S glucocorticoid receptor to the 7-8 S form is dependent on both RNA and protein factors.

The DEAE-cellulose-purified 4 S form of the rat liver glucocorticoid receptor can associate with cytosolic factors, as evidenced by an alteration of the sedimentation value of the 7-8 S form. On the basis of sedimentation profile, this form is indistinguishable from the activated, low-salt 7-8 S form isolated from rat liver cytosol. In addition, both the endogenous and reconstituted 7-8 S receptor can bind DNA as the 7-8 S form. In keeping with our reports that the endogenous form of the 7-8 S receptor is sensitive to RNAase digestion, treatment of the cytosol with RNAase prior to mixing with the 4 S receptor prevents the formation of the 7-8 S material. Moreover, warming the cytosol to 50 degrees C prior to mixing with the 4 S receptor also eliminates the ability to form the heavier material. Since RNA is heat-stable, this suggests that other factors may be involved. Treatment of the cytosol with N-ethylmaleimide, a reagent reported to be specific for sulfhydryl groups, also eliminates 7-8 S generating ability. These observations suggest that a protein may be a component of the 7-8 S generating material. This is substantiated by the observation that trypsin or chymotrypsin treatment of the cytosol mitigates the ability of the cytosol to form the 7-8 S material and results in the appearance of a form of the receptor that sediments at approximately 6 S. Protease treatment of partially purified material eliminates the 7-8 S generating activity entirely. We conclude that the 7-8 S form of the receptor can be reconstituted from the 4 S receptor via association with at least two other cytosolic factors, a protein and an RNA.

Studies with chymotrypsin and RNAase showing a heterooligomeric structure of the glucocorticoid receptor complex from rat liver which is stabilized by a low molecular weight factor.

The glucocorticoid receptor from rat liver displays a differential sensitivity toward digestion by chymotrypsin and RNAase A that is dependent on its activation state. Unactivated (9-10 S) receptor is not digested by these enzymes, while activated 7-8 S receptor is. Chymotrypsin treatment yields an approx. 3 S form, while RNAase treatment yields a 4.9 S form that is distinct from the high-salt 4 S form. To firmly establish that the results are due to specific hydrolytic activities of the particular enzymes, we show that the chymotrypsin effect is inhibited by diisopropylfluorophosphate and not RNAasin, while the reverse is true for RNAase A. We further show that the differential sensitivity toward chymotrypsin is due to the association of a proteinase-resistant, heat-stable low molecular weight factor with the unactivated glucocorticoid receptor. When this factor is removed by warming, dialysis or molecular sieving of the receptor complex, the complex becomes sensitive to chymotrypsin. We also show that moderate chymotrypsin treatment yields a 6-7 S form of the receptor which is composed of, at least, RNA and the 4 S receptor. On the basis of these results, we propose that the 9-10 S receptor is composed of a low molecular weight stabilizing factor whose presence apparently alters the conformation of the complex such that the RNA and the RNA-binding site of the receptor are protected, a chymotrypsin-sensitive factor, RNA and the 4 S receptor itself.