Association of the glucocorticoid hormone receptor with ribonucleic acid

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp9O, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp9O and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp9O. A hormone-dependent dissociation of hsp9O from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp9O in nuclear receptor complexes. In a cell-free system, hsp9O binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23°C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.

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“…receptors that interact with additional, less well characterized proteins (12,37,60) or with RNA (3,17,61).…”

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confidence: 99%

Binding of heat shock proteins to the avian progesterone receptor.

Kost¹,

Smith²,

Sullivan³

et al. 1989

Mol. Cell. Biol.

191

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“…The gel was dried and exposed to an XAR-5 Kodak film using Dupont intensifying screens. To prepare RNA markers, total RNA was extracted from rat hepatoma cells, incubated with [3H]uridine [21] and co-electrophoresed. The small RNAs were identified by comparison with published data [45].…”

Section: Labeling and Identification Of R N Amentioning

confidence: 99%

“…Housley and Pratt [31] demonstrated that RNase removed a 21 000-M, component associated with the purified receptor from mouse L-cells. In addition, it was reported that the transformed receptor from mouse At-T20 cells could bind endogenous tRNA, to give intermediate transformed receptor species of greater size [19, 321. An artefactual association of RNA with the receptor during the preparation of cytosol was unlikely since we had previously demonstrated that the untransformed receptor of rat hepatoma cells behaves as a ribonucleoprotein after crosslinking not only in vitro but also in intact cells [21]. Experiments performed to discriminate between a nonspecific RNA-receptor association in cytosol and the possibility that RNA is an integral component of the untransformed highly purified receptor, have yielded contradictory results [20, 331. On the one hand, it was shown that the 9-S untransformed receptor was RNase-insensitive [33].…”

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confidence: 99%

RNA binding to the untransformed glucocorticoid receptor.

Sablonnière

Economidis²,

Lefèbvre

et al. 1988

European Journal of Biochemistry

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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...

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“…The nuclear skeleton appears to support DNA replication (Pardoll et al, 1980 ;Tubo et al, 1985) and transcription (Robinson et al, 1983 ;Ciejek et al, 1983 ; by tightly anchoring DNP. This structure also binds hnRNP (Herman et al, 1978 ;Miller et al, 1978a ;Van Eekelen and Van Venrooij, 1981) and snRNP (Miller et a/., 1978b ;Gallinaro et al, 1983) Rousseau, 1985). RNA is a potent competitor for the binding of receptor-androgen (Liao et al, 1980), -oestrogen (Feldman et al, 1981 ;Chong and Lippman, 1982), and -dexamethasone (Tymoczko et al, 1982) (Palmiter and Carey, 1974 ;Cox, 1977) ; similarly, oestrogen or progesterone was demonstrated to affect the half-life of coinalbumin mRNA in chick oviduct (Mc Knight and Palmiter, 1979), and androgen to modulate the half-life of prostatic binding protein mRNAs (Page and Parker, 1982).…”

Section: Introductionmentioning

confidence: 99%

“…In this context, receptor-RNA interaction might not only be an important mediator of RNA processing and transport, but also a component of receptor processing and/or transport back to the « cytosol ». Recycling of nuclear steroid-receptors to their cytosolic form nevertheless remains an enigma (Horwitz and Mc Guire, 1980 ;Kasid et al, 1984), yet there is evidence that untransformed « cytosolic receptors » can exist complexed with RNA (Chong and Lippman, 1982 ;Tymoczko and Phillips, 1983 ;Economidis and Rousseau, 1985 …”

Section: Introductionmentioning

confidence: 99%