Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex.

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

Characterization of a second estrogen receptor species in chick oviduct.

Scatchard analysis of estradiol binding to chick oviduct cytosol is consistent with the existence of two high-affinity estrogen receptors which bind this ligand with equilibrium dissociation constants (Kd) of 0.06 and 0.8 mM. While the higher affinity receptor has been characterized and purified [Smith, R. G., & Schwartz, R.J. (1979) Biochem. J. 184, 331-343], the properties of the lower affinity receptor have not previously been defined; thus, its existence has been questioned. We now report the separation of the two receptors by low-capacity affinity chromatography, by sucrose density gradient centrifugation, and the partial separation by diethylaminoethyl ion-exchange chromatography. The former utilizes differences in estrogen binding kinetics associated with each receptor; the rate of dissociation (k-1) of [3H]estradiol from each receptor is identical (k-1 = 7.7 X 10(-5) s-1) while rates of association (k1) reflect the 10-fold differences in Kd. The lower affinity receptor has a k1 = 1.63 X 10(5) M-1 s-1, and the higher affinity receptor has a k1 = 1.33 X 10(6) M-1 s-1. The lower affinity receptor sediments at 3.5 S on sucrose density gradients compared to 4.2 S for the high-affinity form; it has high specificity for estrogen and is tissue specific, and its nuclear occupancy is associated with increases in ovalbumin gene transcription; thus, this macromolecule meets the criteria of an estrogen receptor. Augmentation of this receptor is possible by treatment of the cytosol with ATP/Mg2+, illustrating the existence of a non-steroid binding species.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP mediated receptor interconversion as a model of estrogen action: isolation of the factor which converts the non-estrogen binding form of the receptor to the lower affinity binding form.

Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third non-estrogen binding form of the receptor, designated Rnb, has also been identified. These three forms of the receptor are interconvertible and appear to have a common molecular weight of approx. 66,000 under denaturing conditions. Hydroxytamoxifen binds preferentially and with high affinity to Rx (Kd 0.03 nM) and the conversion of Rx to Ry which is mediated by gamma phosphoryl group of ATP is also inhibitable by hydroxytamoxifen. Thus receptor interconversion, which may have general application to hormone action, potentially explains agonist/antagonist activity. The conversion of the non-estrogen binding form of the receptor (Rnb) to the lower affinity receptor (Ry) in chick oviduct cytosol is catalyzed by a reaction requiring the loss of the terminal phosphoryl moiety from ATP. There is a specific requirement for Mg2+. We now describe that ammonium sulfate fractionation of the cytosol allows the separation of the receptor entities from the "activating factor" (Fy) that catalyzes the conversion of Rnb to Ry. In the presence of gamma [32P]-ATP at 30 degrees C the purified non-steroid binding form of the receptor is phosphorylated. Phosphoamino acid analysis using Partisil-10 SAX anion exchange resin demonstrates that a serine is phosphorylated; and quantitation of the phosphorylation is indicative of one phosphoserine/receptor molecule. Treatment of the receptor with the partially purified activating factor to induce estradiol binding causes a dramatic reduction in phosphorylation.

Receptor interconversion model of hormone action. 1. Purification of a factor involved in conferring estradiol binding properties to the estrogen receptor.

We previously demonstrated that the chick oviduct estrogen receptor exists in three interconvertible forms. Two of these forms bind estradiol with high but distinct affinities. A third form exists as a non-estrogen binding recyclable form, Rnb, which upon treatment with ATP/Mg2+ is quantitatively converted to the lower affinity estradiol binding form. We now describe the isolation from chick oviduct cytosol of a factor involved in this conversion and its 1100-fold purification by ammonium sulfate fractionation, DEAE ion-exchange chromatography, and size-exclusion HPLC. The factor elutes from the size-exclusion column with an apparent molecular weight of 40,000. This highly purified factor potentiates estradiol binding in a dose-dependent manner in the presence of ATP/Mg2+. Its activity is destroyed by heating or by trypsin treatment but is relatively stable to freezing and thawing and is inert to treatment with reducing agents. ATP is an essential nucleotide substrate; GTP and cyclic nucleotides are inactive. Studies of cation dependence demonstrate that Mg2+ is also essential; Ca2+ alone is completely ineffective in catalyzing receptor potentiation and does not synergize with Mg2+. In the presence of excess ATP/Mg2+ and a fixed concentration of Fy, the Km for potentiation of estradiol binding is approximately 0.4 nM.

Reversible activation of non-steroid binding oestrogen receptor.

Two high-affinity oestrogen receptors have been identified in the chick oviduct with equilibrium dissociation constants (Kd) of 0.1 and 1 nM, differing in their binding kinetics, role in ovalbumin synthesis and independent regulation in vivo. The higher-affinity receptor (X) increases RNA polymerase II activity directly, whereas the low-affinity receptor (Y) seems to be necessary to confer specificity to transcription of oestrogen-dependent genes. Acute administration of progesterone to oestrogen-stimulated chicks results in preferential destruction of the nuclear Y receptor accompanied by interruption of ovalbumin gene transcription. Here we demonstrate that receptor Y exists in a non-oestradiol binding form (Ynb) which can be activated to the binding form in vitro by treatment with either ATP or ADP. Furthermore, dialysis of oviduct cytosol, which has no effect on the high-affinity receptor X, converts receptor Y to Ynb; receptor Y can then be recovered by treatment with ATP in the presence of Mg2+ and independently of Ca2+. This is the first report of the controlled interconversion between a non-steroid binding form of oestrogen receptor and active receptor in a tissue that contains two independently regulated oestrogen receptor types.

Receptor interconversion model of hormone action. I. ATP-mediated conversion of estrogen receptors from a high to lower affinity state and its relationship to antiestrogen action.

We have previously reported that the estrogen receptor exists in three distinct states in the oviduct of the estrogen-treated chick. Since two of these forms bind estrogen and possess a number of similar properties, the intrinsic relationship between these two receptor forms has been investigated. We now show that a quantitative conversion of the high affinity (Rx) to the lower affinity (Ry) state can be induced by mild heating (30 degrees C) in the presence of estradiol and ATP, or the synthetic analogue alpha,beta-methylene adenosine triphosphate and the divalent cation Mg2+. Other nucleotides, including ADP, GTP, CTP, cAMP, and cGMP, as well as the nonhydrolyzable analogues beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate are ineffective. The conversion occurs only partially in the absence of estradiol but completely in its presence. The process is not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride and alpha 2-macroglobulin, and when conversion is induced by low concentrations of ATP (1 mM), a time dependent reequilibration back to Rx occurs. These observations and the fact that the pure hormone-binding peptides Rx and Ry have similar molecular weights on sodium dodecyl sulfate-polyacrylamide gels (66,000) confirm that proteolysis is not involved in the conversion. Moreover their physical properties suggest that Rx and Ry exist in alternate conformations, with Ry being favored as a result of an ATP-mediated event involving the gamma-phosphoryl moiety. The biological relevance of the receptor conversion is suggested by studies with the antiestrogen hydroxytamoxifen. This antiestrogen binds to Rx with higher affinity than either estradiol or diethylstilbestrol but with low affinity to Ry. Hydroxytamoxifen also inhibits the conversion of Rx to Ry. Since this antiestrogen is a complete antagonist in the chick oviduct and prevents estradiol-induced stimulation of ovalbumin gene transcription, it is speculated that Rx to Ry conversion is crucial for ovalbumin gene activation and that Rx may act as a transcriptional repressor. Furthermore, since Rx and Ry both bind to nuclei and DNA, it is proposed that the presence of Ry is a better predictor of ovalbumin gene activation than DNA binding alone.

Detoxications in peripatus. Sulphate, phosphate and histidine conjugations.

Phenols were detoxified in the Onycophoran Peripatoides novaezealandiae by conjugation with sulphuric acid and phosphoric acid, but no evidence for a glycoside detoxication could be found. [(14)C]Benzoic acid was metabolized in 24h to N(2)-benzoyl-l-histidine, which was identified by electrophoresis, chromatography and dilution analysis. Similar conjugates were formed with p-aminobenzoic acid and p-nitrobenzoic acid. In longer-duration experiments further unidentified metabolites were formed, two of which appeared to result from the further metabolism of the histidine conjugate.

Receptor interconversion model of hormone action. 2. Requirement of both kinase and phosphatase activities for conferring estrogen binding activity to the estrogen receptor.

Three interconvertible forms of the estrogen receptor have been identified in the oviduct of estrogen-stimulated chicks. The non-estradiol binding form (Rnb) can be converted to the lower affinity binding form (Ry, Kd = 0.8 nM) by a process requiring the gamma-phosphoryl moiety of ATP. The enzymatic activity (Fy) essential for this "receptor potentiation" has been isolated from oviduct cytosol using ammonium sulfate fractionation, DEAE chromatography, and HPLC size-exclusion chromatography. The potentiation appears to require both kinase and phosphatase activities. The Fy kinase characteristically phosphorylates casein, histones, and glycogen synthase. Comparison of the kinase with casein kinase II, which also phosphorylates casein and glycogen synthase, indicates that Fy represents a distinct protein kinase since its activity is not stimulated by spermine or inhibited by heparin. Fy-mediated conversion of Rnb to Ry is blocked by the phosphatase inhibitors vanadate, fluoride, and pyrophosphate. The substrate specificity of the Fy phosphatase activity is distinct from that of the two well-characterized protein phosphatases 1 and 2A. Moreover, the requirement for Fy phosphatase activity in converting Rnb to Ry could not be mimicked by its substitution with purified protein phosphatases 1 or 2A. The unique substrate specificity of the oviduct protein phosphatase and protein kinase, which are apparently necessary to confer estradiol binding characteristics to the receptor, implies that these enzymes play a key role in the control of the estrogen receptor in its function as a transcription factor.

Receptor interconversion model of hormone action. II. Nucleotide-mediated conversion of estrogen receptors from nonsteroid binding to the lower affinity binding state.

Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third nonestrogen binding form of the receptor, designated Rnb, is now described which exists in short-term estrogen withdrawn chick oviduct cytosol. A model is presented in which the receptor can be interconverted between the three states. The interconversion is monitored by Scatchard analysis, sucrose density gradient analysis, and affinity labeling using [3H]tamoxifen aziridine followed by receptor purification with estrogen receptor monoclonal antibody affinity chromatography and sodium dodecyl sulfate-gel electrophoresis. The results are consistent with each state existing in different conformations having a common molecular weight of approximately 66,000. This paper defines the conditions and nucleotide requirements for the Rnb to Ry conversion. The conversion to the steroid binding form is induced by ATP, ADP, and GTP. Cyclic nucleotides are ineffective. There is a specific requirement for Mg2+; neither Ca2+ nor Mn2+ will substitute. Nonhydrolyzable nucleotide analogues were tested for their relative efficiency to convert Rnb to Ry. Conversion occurred with alpha,beta-methylene adenosine triphosphate, but beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate were inert. Thus, activation of Rnb to form Ry appears to be catalyzed by an event requiring the loss of the terminal phosphoryl moiety from either ATP or ADP. Receptor derived from conversion of Rnb to Ry has the same physical properties as native Ry. Activation of Rnb is to Ry specifically; no increase in the Rx form of estrogen receptor was ever observed. The accompanying paper similarly describes the Rx to Ry conversion. Since these data also explain observations made with glucocorticoid and with epidermal growth factor receptors, it is speculated that the receptor interconversion model may have general application to hormone action.