THE 2 NATIVE ESTROGEN-RECEPTOR FORMS OF 8S AND 4S PRESENT IN CYTOSOL FROM HUMAN UTERINE TISSUES DISPLAY OPPOSITE REACTIVITIES WITH THE ANTIESTROGEN TAMOXIFEN AZIRIDINE AND THE ESTROGEN-RESPONSIVE ELEMENT

We have investigated the capability of the different native ER forms, present in cytosols from human uterine tissues, of reacting with the a ntiestrogen [H-3]Tamoxifen aziridine ([H-3]TA) and with the Estrogen R esponsive Element (ERE). Cytosols from uterine leiomyoma (myoma) prepa red in buffer containing 40 mM molybdate and protease inhibitors, labe lled with [3H]estradiol and analyzed in low-salt sucrose gradient show ed 8S and 4S ER forms. The same cytosols labelled with [H-3]TA only sh owed a 4S ER form, whereas the ERE only reacted with fractions from th e 8S peak. The band of ERE reaction in the EMSA assay showed a lower r elative mobility than the band labelled with [3H]TA, but both bands co ntained immunoreactive ER of 65 kDa. Electrophoretic mobility of the [ H-3]TA-labelled band in that system was not affected by cytosol treatm ent with cross-linkers or SDS, which suggests that it is a monomeric p rotein. The [H-3]TA-binding 4S ER form was found in all studied myoma samples, as well as in human endometrium or myometrium, but not in rat tissues. These results suggest that the 8S and 4S ER form were alread y present before cytosol from human uterine tissues comes into contact with the molybdate buffer. They both contain the same ER molecule of 65 kDa, either in the free form or as an oligomer. Only the ER dimers, which have been described both in the cytosolic 8S form and in the nu clear 4-5S form, react with the ERE. [H-3]TA only binds to the 4S ER m onomer probably because its binding site is concealed in the 8S form u nder these experimental conditions. The opposite reactivity of the 8S and 4S ER forms with [3H]TA and the ERE support the hypothesis that th ey may constitute separate entities with a different physiological rol e. (C) 1998 Elsevier Science Ltd. All rights reserved.