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

Abstract:

: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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Fritsch M,Aluker M,Murdoch FE

doi

10.1021/bi982190k

subject

Has Abstract

pub_date

1999-06-01 00:00:00

pages

6987-96

issue

22

eissn

0006-2960

issn

1520-4995

pii

bi982190k

journal_volume

38

pub_type

杂志文章