Identification of a unique liganded estrogen receptor complex released from the nucleus by decavanadate.
Unoccupied estrogen receptor (ER) can be extracted from tissues by homogenization with a hypotonic buffer, whereas hormone-occupied ER becomes tightly bound to the nuclear pellet and must be extracted with high-salt-containing buffers. The molecular basis for estrogen-induced tight nuclear binding of ER remains an important puzzle. The different subcellular fractionation behaviors of the occupied and unoccupied ER are presumed to be due to a difference in their ability to interact with nuclear components, such as DNA and proteins. The proteins that are the targets for interaction with the hormone-occupied ER may be important for transcriptional regulation. However, the salt-extracted ER is recovered as a homodimer, and associated proteins are presumably lost due to the high-salt conditions. We have discovered an alternate 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 and DNA binding and is full-length by western blot analysis. Our data suggest that the mechanism of ER release is by decavanadate competition with nuclear DNA, rather than by inhibition of a phosphotyrosine phosphatase. Of particular interest, the decavanadate released occupied ER complex shows distinct behavior by sucrose density gradient sedimentation analysis. It is larger than the salt-extracted transformed ER, suggesting that an occupied ER in complex with nuclear proteins may be released from the nucleus by decavanadate.
Fritsch
M
M
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Aluker
M
M
Murdoch
F E
FE
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D012492:Salts; D014325:Tromethamine; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology*
D008024:Ligands; D046911:Macromolecular Substances; D011960:Receptors, Estrogen; D012492:Salts; D014325:Tromethamine; D014638:Vanadates; D004247:DNA; D010749:Phosphoprotein Phosphatases