HSP70 IS NOT REQUIRED FOR HIGH-AFFINITY BINDING OF PURIFIED CALF UTERINE ESTROGEN-RECEPTOR TO ESTROGEN RESPONSE ELEMENT DNA IN-VITRO

Bovine estrogen receptor (ER) was purified to near homogeneity by estr ogen response element (ERE) affinity chromatography, and its ERE bindi ng ability was measured in vitro. Highly purified ER bound EREs with r educed affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whethe r addition of purified recombinant human hsp70 or purified bovine hsp7 0 would restore the higher ERE binding affinity, stoichiometry, and li gand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE bind ing was not affected by antibodies to either constitutive or induced f orms of hsp70, regardless of ER purity. Addition of purified hsp70, wi th or without ATP and Mg2+, did not affect the association or dissocia tion rates of highly purified liganded ER binding to ERE. hsp70 Did no t alter the total amount of ER-ERE complex formed. Similarly, hsp70 di d not affect the rate of [H-3]estradiol (E-2) or [H-3]4-hydroxytamoxif en (4-OHT) ligand dissociation from ER in the presence or absence of E REs. These data contrast with a report showing that maximal ERE bindin g by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its pro per tertiary structure, and hsp70 does not impact ER ligand binding do main conformation. (C) 1997 Elsevier Science Ltd. All rights reserved.