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.

A comparative study of antiestrogen action: temporal patterns of antagonism of estrogen stimulated uterine growth and effects on estrogen receptor levels.

Studies were undertaken to ascertain the effects of structural modification of two well-known antiestrogens (CI-628 and U-11,100A) on their estrogenic and antiestrogenic potencies and temporal patterns of effectiveness in the immature rat uterus. Changes in the chemical structures of these anti-estrogens produce compounds with markedly different affinities for the uterine estrogen receptor as measured in an in vitro cell-free cytosol system; binding affinities relative to estradiol (100%) are: CI-628, 4%; CI-680, 34%; 94X1127 (94X),222%; U-11,100A (UA), 6%; and U-23,469 (U-23), 0.1%. Although all five antiestrogens (daily injections of 50 microng over three days) appear equally effective in stimulating 72 h uterine weight when given alone, or in blocking the estradiol-stimulated weight increase when given with estradiol, marked differences in their potencies are noted when the effects of the compounds are monitored beyond 24 h following a single injection. The compounds CI-628, CI-680 and UA (50 microng sc in saline), which have a methylated hydroxyl group (at the site analogous to the steroid position 3), show a prolonged maintenance of elevated levels of nuclear receptor (beyond 48 h) and elevated uterine weight (until 72 h); this correlates with a prolonged period of depressed cytoplasmic receptor levels (beyond 48 h) and prolonged uterine insensitivity to estrogen (beyond 36 h as monitored by 3 h wet weight response). In contrast, a single injection of 50 microng of 94X (having a free hydroxyl group) or U-23 (with a side chain and central ring different from UA) maintained nuclear receptor levels elevated for only 12 h (94X) or 36 h (U-23) and uterine weights declined after 36-48 h; cytoplasmic receptor levels remained depressed for only 12 h (94X) or 24 h (U-23) and then returned to control levels or above by 36 h. These latter compounds likewise evoked the shortest period of uterine insensitivity to estrogen (ineffective as antagonists by 36 h). Comparative studies with these five compounds indicate that they are effective as estrogen antagonists only so long as they maintain cytoplasmic receptor levels low, and that the magnitude of the responsiveness to estradiol after antiestrogen correlates with the extent of reappearance of cytoplasmic receptor. Thus, chemical modifications of antiestrogen structure have significant effects on their temporal patterns of effectiveness as estrogens and as estrogen antagonists.

Desmethylnafoxidine aziridine: an electrophilic affinity label for the estrogen receptor with high efficiency and selectivity.

Desmethylnafoxidine aziridine (Naf-Az), an affinity label for the estrogen receptor based structurally on the antiestrogen nafoxidine, has been prepared in unlabeled and in high specific activity, tritium-labeled form and has been evaluated for its apparent competitive binding, and time-dependent irreversible, covalent attachment to the estrogen receptor. Naf-Az was synthesized through a key 1,2-diaryl-3,4-dihydronaphthalene intermediate that was prepared from 6-methoxy-1-tetralone by two routes involving alternate strategies for arylation. Conversion of the diaryldihydronaphthalene to Naf-Az through a series of deprotection-activation reactions culminated in ethyleneimine displacement of a methanesulfonate. The tritium-labeled material was prepared by tritium-iodine exchange on an iodinated methanesulfonate precursor, followed by ethyleneimine displacement. Compared to our previously-prepared reagent tamoxifen aziridine (Tam-Az), Naf-Az has a higher apparent competitive binding affinity, and it reacts with the estrogen receptor in cytosol preparations and in intact MCF-7 breast cancer cells rapidly and with at least comparable efficiency and selectivity. SDS-polyacrylamide gel electrophoretic analysis confirms its selective labeling of the Mr 66,000 estrogen receptor. Naf-Az should prove to be useful in studies aimed at characterizing the properties and structure of estrogen receptors.

Multiple DNA-binding estrogen receptor forms resolved by interaction with immobilized metal ions. Identification of a metal-binding domain.

Immobilized metal ions have been used to characterize and locate metal ion-specific binding domains on the surface of the DNA-binding form of the estrogen receptor protein. Soluble estrogen receptors in calf uterine cytosol were labeled with [3H]estradiol and transformed to the DNA-binding configuration by brief exposure (30 min) to 3 M urea at 0-4 degrees C. The transformed receptors were purified in the presence of 3 M urea using single-stranded calf thymus DNA-agarose and characterized by high-performance size-exclusion chromatography (Stokes radius of 7.0-7.5 nm) and sucrose density gradient centrifugation (4.25 S) as dimers of 130,000 Da. Such receptor preparations subsequently labeled with [3H]desmethylnafoxidine aziridine by ligand exchange revealed one major peak of radioactivity (67 kDa) by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis. When analyzed by immobilized metal ion affinity chromatography on iminodiacetate (IDA)-agarose loaded with Cu(II), Ni(II), or Zn(II) ions, the receptor was bound with various degrees of affinity and metal interaction heterogeneity even in the presence of 0.5 M NaCl to neutralize electrostatic interactions. The intact DNA-binding receptor dimers were most tightly bound to IDA-Cu(II) and IDA-Ni(II), but were eluted with 100-200 nM imidazole. The receptors were bound less tightly to IDA-Zn(II), and four separate peaks of receptor activity were resolved by elution with 10, 15, 30, and 100 mM imidazole (n = 27). Limited trypsin digestion of the DNA-binding receptor forms resulted in the generation of a 2.8-nm fragment with both the DNA-binding and metal-binding domains removed or destroyed. These results demonstrate that DNA-binding estrogen receptor dimers have high affinity metal ion-binding sites which are located at the DNA-binding domain. We have found (Zn(II) interaction chromatography to be unique thus far in its ability to resolve separate DNA-binding receptor forms.