Binding of the estradiol receptor from calf uterus to the chromatin: Active forms

Sala‐Trepat, José M.; Vallet-Strouve, C.

doi:10.1016/0005-2795(74)90168-8

Cited by 32 publications

(6 citation statements)

References 25 publications

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“…Problems with the receptor titration were not anticipated since most reports concerning artifiacts in this approach utilized crude cytosol, whereas a more purified receptor preparation was used in the studies in this paper. It has been reported that the partially purified (ammonium sulfate precipitated) estrogen receptor from calf uterus seemed to lack the nuclear binding inhibitor apparent in the crude cytosol receptor preparation (Sala-Trepat & Vallet-Strouve, 1974). Since the binding of [3H]progesterone to NAP requires intact receptor, a variety of assay conditions used for blanks (e.g., assays with no DNA or NAP and assays with no receptor or denatured receptor) resulted in only a minor contribution to the total radioactivity in each of the assays.…”

Section: Discussionmentioning

confidence: 99%

Factors affecting the binding of chick oviduct progesterone receptor to deoxyribonucleic acid: evidence that deoxyribonucleic acid alone is not the nuclear acceptor site

Thrall

Spelsberg²

1980

Biochemistry

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Studies on the interaction of the chick oviduct progesterone-receptor complex (P-R) with various nuclear components revealed a variable, nonsaturable binding of P-R to pure deoxyribonucleic acid (DNA). In contrast, a receptor-dependent, saturable, high level of binding of P-R was observed with a nonhistone protein-DNA complex called nucleoacidic protein (NAP). Three categories of factors were identified which affected the binding of P-R to the DNA. These were (1) the conditions of the binding assay, (2) the properties of the receptor, and (3) the state of the DNA. The conditions in the binding assay which affect DNA binding are the choice of the blanks, the salt concentration, and the pH of the assay. The receptor preparations display their own characteristic levels of binding to native DNA. The basis of this DNA binding capacity by each preparation is unknown. Lastly, the purity and the integrity of the DNA itself determine the level of binding of the P-R. Protein impurities, moderate degradation of the DNA by enzymatic or physical fragmentation, and ultraviolet (UV) light treatment greatly enhance the receptor binding to the DNA. The extent of binding to DNA depends on the degree of damage. Interestingly, totally denatured (single-stranded) DNA displays little or no binding of the P-R. Seasonal differences which are observed for the binding of P-R to chromatin in vivo and in vitro and to NAP in vitro do not occur with DNA whether it is undamaged or damaged. It is concluded from these studies that under controlled conditions and by using DNA preparations as native as possible, minimal binding of P-R to pure DNA occurs. The numerous reports in the literature describing marked binding of the steroid-receptor complex to DNA may well be due to conditions described in this paper. Further, it is concluded that native or partially degraded DNA alone does not appear to represent the native nuclear acceptor sites for the chick oviduct P-R. In contrast, the DNA-nonhistone protein (acceptor protein) complexes do show characteristics of the native-like acceptor sites.

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Section: Discussionmentioning

confidence: 99%

Factors affecting the binding of chick oviduct progesterone receptor to deoxyribonucleic acid: evidence that deoxyribonucleic acid alone is not the nuclear acceptor site

Thrall

Spelsberg²

1980

Biochemistry

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“…The great sensitivity of steroid receptors to proteolytic attack is well-known (24). Even though limited proteolysis of receptor, due to endogenous or exogenous enzymes, may lead to functionally active forms capable of binding to nuclei or DNA (25)(26)(27), receptor proteolysis has been generally implicated in the degradation rather than in the transformation of receptor. One of the conceptual obstacles to accepting proteolysis as the activat- ing step was the fact that no proteolytic enzyme so far studied (28)(29)(30) was able to distinguish between the occupied and the unoccupied receptor.…”

Section: Discussionmentioning

confidence: 99%

Estradiol receptor has proteolytic activity that is responsible for its own transformation.

Puca¹,

Abbondanza²,

Nigro³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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We have investigated the effect of various protease inhibitors and substrates on the hormone-and temperature-dependent binding of partially purified estradiol-receptor complex to isolated nuclei. Only serine protease substrates and inhibitors significantly depressed estradiol receptor transformation. At 20°C, we observed 50% inhibition with about 3 IAM aprotinin or with 1.4 mM dilsopropyl fluorophosphate. Aprotinin also blocked those size and charge modifications of receptor that are characteristic of the transformation process. The estradiol receptor was able to bind to aprotininagarose only under transforming conditions; i.e., the interaction was hormone-and temperature-dependent and inhibited by molybdate. Diisopropyl fluorophosphate, a covalent reagent for serine esterases, competitively inhibited the binding and specifically eluted the estradiol-receptor complex that had been bound to aprotinin-agarose. These results indicate that estradiol receptor transformation is due to the effect of a serine protease and that the receptor itself is endowed with this catalytic activity, which is triggered by the steroid.Transformation of steroid receptors has been widely studied in cell-free systems and has been defined as that process which confers affinity to the steroid-receptor complex for nuclei and DNA. In all systems so far investigated, transformation is accompanied by a decrease in the molecular size and the net electric charge of the receptor. Receptor transformation is an irreversible first-order reaction, inhibited by millimolar concentration of molybdate, and is triggered by the specific steroid (for review, see refs. 1-3). Evidence for the physiological role of this reaction (4-7) supports the theory that transformation is the critical step in steroid hormone action. Numerous mechanisms have been proposed to explain the molecular basis ofthis event, but none has been definitively accepted.We report here that the receptor-transforming activity of exogenous alkaline phosphatase is due to a contaminating proteolytic activity of the enzyme preparation, that serine protease inhibitors and substrates impair transformation of the estradiol receptor, and that estradiol receptor acquires upon transformation a serine binding site for aprotinin. Therefore, we suggest that estradiol receptor transformation is due to the action of a serine protease activated by the hormone and that the catalytic protein is the hormone receptor itself. MATERIALS AND METHODS[2,4,6,7-3H]Estradiol-17P Ci/mmol; 1 Ci = 37 GBq) was from New England Nuclear. Nonradioactive estradiol-17,B was from Calbiochem. Antipain dihydrochloride, soybean trypsin inhibitor (SBTI), pepstatin A, trypsin inhibitor type 11-0 (ovomucoid) from chicken egg white, leupeptin hemisulfate, a1-antitrypsin, tryptophan methyl ester (TrpOMe), phosphoramidone, phenylmethylsulfonyl fluoride, aprotinin, calf intestinal alkaline phosphatase types I-S and VII-S, and p-nitrophenyl phosphate were from Sigma. Nbenzoyl-L-arginine methyl ester (Bz-Arg-OMe), diisopropyl flu...

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“…The nonrandom affinities for polyand oligonucleotides suggest a complimentary configuration on the receptor protein which functions as a recognition site. The separateness of the polynucleotide recognition site from that of steroid binding in E2R, as well as other steroid receptor complexes, was demonstrated by the effects of limited proteolysis (9)(10)(11). Under these conditions, DNA binding was destroyed without affecting retention of the steroid ligand.…”

Section: Introductionmentioning

confidence: 99%

Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins

et al. 1979

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During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.

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Binding of the estradiol receptor from calf uterus to the chromatin: Active forms

Cited by 32 publications

References 25 publications

Factors affecting the binding of chick oviduct progesterone receptor to deoxyribonucleic acid: evidence that deoxyribonucleic acid alone is not the nuclear acceptor site

Factors affecting the binding of chick oviduct progesterone receptor to deoxyribonucleic acid: evidence that deoxyribonucleic acid alone is not the nuclear acceptor site

Estradiol receptor has proteolytic activity that is responsible for its own transformation.

Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins

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