In Vitro Formation of Estrogen Receptor-Heat Shock Protein 90 Complexes

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We previously showed that the 9S estrogen receptor can be reconstituted from purified vero ER (estradiol binding subunit) and purified hsp 90 (heat shock protein 90) in vitro [Inano, K. et al. (1990) FEBS Lett. 267, 157-159]. In this study, we further characterized our reconstitution system to investigate the mechanism underlying the formation of 9S ER. When a vero ER preparation stored at 4°C for more than 20h after affinity chromatography was used for the reconstitution of 9S ER, 0.5M NaSCN was essential, but not Na₂MoO₄ or other reagents. When, however, vero ER was used within 3h after dissociation from an affinity resin, 9S ER could be reconstituted in a relatively high yield without NaSCN. Moreover, if such a fresh vero ER preparation was used, 9S ER could be reconstituted in the absence of NaSCN from not only unoccupied vero ER but also the occupied form. From these results it was suggested that the conformation of purified vero ER tends to change quickly in a time dependent manner, and so a chemical perturbant, NaSCN, is generally necessary for the reconstitution of 9S ER from purified vero ER and purified hsp 90. The concentration of hsp 90 required for the reconstitution was only about 1.0μM, which was lower than its physiological concentration. Based on these results, the mechanism underlying the forma tion of 9S ER was discussed.