Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor

Abstract

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.

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

“…This is inferred from the fact that the catalytically inactive RNase S-protein promotes DNA binding by receptors, both in our system (Tables 4 and 5) and in the purified receptor system of Schmidt et al [18]. At least, it can be said that the effect of the RNase preparation in our system is unrelated to the presumed catalytic effects of RNase on the size and DNA-binding activity of glucocorticoid receptors that have been reported in rat liver cytosol and other systems [20,21 ].…”

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

“…binding moiety f reviewed in Vedeckis (1985)]. Our laboratory has performed a detailed series of studies to elucidate the structure of the glucocorticoid receptor (GC-R)1 *in the mouse AtT-20 pituitary tumor cell line (Vedeckis, 1981(Vedeckis, , 1983aEastman-Reks et al, 1984;Reker et al, 1985;Kovacic-Milivojevic et al, 1985;Vedeckis et al, 1985). We 1 Abbreviations: 94K, 24K, etc., M, of 94000, 24000, etc.…”

Investigation of protein structure by means of 19F‐NMR

“…These and earlier data suggest that an RNA-binding site exists on the receptor, which could interact with small RNAs, both in its 'intermediate transformed form' [19] or in its untransformed form [20]. Whether these RNA-receptor complexes have a role in the cells remains to be demonstrated.…”