Identification of a unique liganded estrogen receptor complex released from the nucleus by decavanadate

Unoccupied estrogen receptor (ER) can be extracted from tissues by homogeni zation with a hypotonic buffer, whereas hormone-occupied ER becomes tightly bound to the nuclear pellet and must be extracted with high-salt-containin g buffers. The molecular basis for estrogen-induced tight nuclear binding o f ER remains an important puzzle. The different subcellular fractionation b ehaviors of the occupied and unoccupied ER are presumed to be due to a diff erence in their ability to interact with nuclear components, such as DNA an d proteins. The proteins that are the targets for interaction with the horm one-occupied ER may be important for transcriptional regulation. However, t he salt-extracted ER is recovered as a homodimer, and associated proteins a re presumably lost due to the high-salt conditions. We have discovered an a lternate method of releasing the occupied ER from the nucleus. Inclusion of 2 mM orthovanadate, polymerized primarily to decavanadate, in a hypotonic buffer efficiently releases over 90% of estrogen-bound ER from the nuclear pellet. The recovered ER complex is fully functional in terms of estrogen a nd DNA binding and is full-length by western blot analysis. Our data sugges t that the mechanism of ER release is by decavanadate competition with nucl ear DNA, rather than by inhibition of a phosphotyrosine phosphatase. Of par ticular interest. the decavanadate released occupied ER complex shows disti nct behavior by sucrose density gradient sedimentation analysis. It is larg er than the salt-extracted transformed ER, suggesting that an occupied ER i n complex with nuclear proteins may be released from the nucleus by decavan adate.