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.
Hutchens
T W
TW
Department of Obstetrics and Gynecology, Baylor College of Medicine, Texas.
McNaught
R W
RW
Yip
T T
TT
Suzuki
T
T
Li
C M
CM
Besch
P K
PK
D000818:Animals; D001388:Aziridines / Q000378:metabolism*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D004965:Estrogen Antagonists / Q000378:metabolism; D005260:Female; D008024:Ligands; D009256:Nafoxidine / Q000031:analogs & derivatives / Q000378:metabolism*; D011759:Pyrrolidines / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology*; D013696:Temperature; D014170:Transformation, Genetic / Q000187:drug effects; D014316:Tritium; D014508:Urea / Q000494:pharmacology*; D014599:Uterus / Q000378:metabolism / Q000648:ultrastructure
D001388:Aziridines; D004965:Estrogen Antagonists; D008024:Ligands; D011759:Pyrrolidines; D011960:Receptors, Estrogen; D012492:Salts; D014316:Tritium; C053702:desmethylnafoxidine aziridine; D009256:Nafoxidine; D004958:Estradiol; D014508:Urea; D004247:DNA