Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes

Schmidt, Thomas J.; Diehl, Edward E.; Davidson, Craig; Puk, Michael J.; Webb, Maria L.; Litwack, Gerald

doi:10.1021/bi00368a018

Cited by 19 publications

(12 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There are reports of a factor similar to the one that we describe here which is capable of converting highly purified unactivated 9-10 S glucocorticoid receptor directly into the 4 S form (Schmidt et al, 1985(Schmidt et al, , 1986. Those workers suggest that the factor is RNAase, because RNAase A can substitute for the factor.…”

Section: Discussionmentioning

confidence: 56%

“…It is not clear why RNAase A has no effect on the partially purified 9-10 S receptor used in these studies while having a dramatic effect on the highly purified 9-10 S receptor. Those workers did note that actual RNA hydrolysis by the RNAase is not required for activation stimulation to occur, but an intact RNAbinding site on the RNAase is (Schmidt et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Tymoczko

Ahern

Unger

et al. 1988

Biochemical Journal

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 99%

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

Tymoczko

Ahern

Unger

et al. 1988

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Schmidt et al [25] suggested that an RNA-binding site is present in the receptor and that a small RNA molecule associated with the receptor was lost upon transformation. Still, the role of an endogenous cytosolic RNase in receptor transformation remains controversial.…”

Section: Discussionmentioning

confidence: 99%

“…It was reported that RNA could be one of these putative 'receptor-binding factors' as suggested by studies on the glucocorticoid [19 -211, oestrogen and progesterone receptors [22, 231. RNase A reportedly converts the oligomeric 9-S form of the glucocorticoid receptor to a 4-S monomeric species with a concomitant increase in DNA binding [24, 251. Indeed, Schmidt et al [25] have demonstrated that RNase A stimulates the DNA-binding ability of purified rat glucocorticoid-receptor complexes in a dose-dependent manner. Cytosolic glucocorticoid-receptor complexes can bind to polynucleotides, and binding of the receptor to DNA-cellulose can be displaced by RNA [26,271.…”

mentioning

confidence: 99%

RNA binding to the untransformed glucocorticoid receptor.

Sablonnière

Economidis²,

Lefèbvre

et al. 1988

European Journal of Biochemistry

View full text Add to dashboard Cite

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (R,) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate. In contrast, the highly purified glucocorticoid receptor yielded a sharp symmetrical peak of R, = 7.1 nm. We demonstrate that the 7.1-nm component could not result from a proteolytic degradation of the 8.3-nm receptor form. The same receptor heterogeneity was observed in thymus cytosol which contains less proteases than liver. After labeling with [3H]dexamethasone 21 -mesylate and SDSjPAGE the same 94-kDa receptor band was revealed in both the 8.3-nm and 7.1-nm forms. Immunoblotting experiments showed that both the 94-kDa hormone-binding subunit and the 90-kDa heat-shock protein were present in the two different receptor forms. The 8.3-nm receptor form was converted to the 7.1-nm receptor form after treatment by ribonuclease A in the presence of molybdate and this effect was dose-dependent, being completely prevented by placental ribonuclease inhibitor (RNasin). In contrast, in the presence of molybdate, the 7.1-nm receptor form was ribonuclease-insensitive. Treatment of cytosol with RNase A in the absence of molybdate, partially shifted the untransformed receptor towards the 5.2-nm transformed receptor form. This effect was abolished by placental ribonuclease inhibitor. RNase S protein, an enzymatically inactive proteolytic fragment of RNase A, or S1 nuclease, which is specific for single-stranded nucleic acids, were ineffective when used instead of RNase A. In contrast, cobra venom endonuclease, which preferentially attacks double-stranded regions of small RNAs, caused a complete conversion of the 7 -8-nm untransformed receptor to the 5.2-nm transformed receptor form. These results were not observed in the presence of molybdate. Addition of RNasin prior to heating cytosol in the absence of molybdate did not prevent the receptor from dissociating to the 5.2-nm form, suggesting that an endogenous RNase is not involved in the transformation process. The 7.1-nm receptor form was shifted to a 9.2-nm complex when incubated with an excess of GR 49 antireceptor antibody, whereas the 8.3-nm receptor form did not bind to the antibody. Furthermore, both these receptor forms were shifted when incubated with the monoclonal AC 88 anti-(heat-shock protein) antibody. These results domonstrate that a ribonuclease-sensitive cytosolic factor is closely associated to the receptor protein inside the heteromeric untransformed glucocorticoid receptor. To identify the receptor-associated RNA, the untransformed receptor was purified by protamine sulfate precipitation, affinity chromatography and size-exclusion HPLC. Samples were extracted with phenol and RNA was end-labeled with 32P. Receptor purification led to a specific enrichment in a 120-nucleotide RNA band which could not be detected when a mock purification was performed. Taken together these results suggest tha...

show abstract

“…Schmidt et al [18] have recently shown that RNase incubated for 1 h on ice with 3.5 mM MMTS and filtered through Sephadex G.50. Additions of endogenous activating factor (EAF, 40% by volume, final protein concentration 0.68 mg/ml), RNase A (0.68 mg/ml), an equimolar amount of RNase S-protein, or S-peptide, or each of these preincubated for I h at room temperature with 20 mM DTT were made as indicated, and the mixtures were incubated on ice for 1 h prior to assay of steroid binding and binding to DNA-cellulose.…”

Section: Rnase Also Stimulates Dna Bindingmentioning

confidence: 99%

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease

Tienrungroj

Pratt

Grippo

et al. 1987

Journal of Steroid Biochemistry

View full text Add to dashboard Cite

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).

show abstract

Effects of bovine pancreatic ribonuclease A, S protein, and S peptide on activation of purified rat hepatic glucocorticoid-receptor complexes

Cited by 19 publications

References 44 publications

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

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

RNA binding to the untransformed glucocorticoid receptor.

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease

Contact Info

Product

Resources

About