Heat Shock Protein 90 Strongly Stimulates the Binding of Purified Estrogen Receptor to Its Responsive Element

We previously showed that the 9 S estrogen receptor (ER) could be reconstituted from purified ER and purified heat shock protein 90 (hsp 90). So, we investigated the role of hsp 90 in the binding of purified ER to an estrogen responsive element (ERE) by using the reconstitution system. ER purified from calf uterus showed a very low binding capacity to an ERE from the vitellogenin A2 gene in the gel mobility shift assay. However, the binding was strongly stimulated by reconstitution with hsp 90 and was proportional to the amount of reconstituted 9 S ER. Hsp 70, a typical molecular chaperone and a component of some steroid receptors, did not cause similar stimulation. The equilibrium dissociation constants (IQ) of the occupied and unoccupied 9 S ER for the ERE were the same as each other, indicating that the binding of ER to the ERE was independent of the ligand. H222, a monoclonal antibody which binds to the hormone-binding domain (HBD) of ER, recovered the high afiinity ER-ERE binding. The binding of hsp 90 to ER suppressed the Triton X-100 stimulated estradiol-dissociation from the ER. The sedimentation coefficients and Stokes' radii of the purified and unpurified cytosolic ER were compared, and it was shown that the purified ER was not unfolded and had a rather compact structure, similar to the cytosolic ER. From these results and others, the mechanism underlying stimulation of the ER-ERE binding by hsp 90 was speculated to be as follows: i) purified ER exists in a molten globule state, ii) hsp 90 recognizes the state and binds to the HBD, and iii) this binding changes the ER to a native form, and thus makes possible the binding of ER to the ERE.