The N-terminal adenosine triphosphate binding domain of Hsp90 is necessaryand sufficient for interaction with estrogen receptor

To understand how the molecular chaperone Hsp90 participates in conformatio nal maturation of the estrogen receptor (ER), we analyzed the interaction o f immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was e ither immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp9O fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain s pecifically ER, similarly to immunoadsorbed Hsp9O. When cells were treated with estradiol and the hormone treatment was maintained during cell homogen ization, binding, and washing steps, GST.90 still interacted efficiently wi th ER, suggesting that ER may form complexes with Hsp90 even after its acti vation by hormone and salt extraction from nuclei. The GST.90-ER interactio n was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously sta bilized by molybdate, indicating that GST.90-ER complexes behave like cytos olic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other c haperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being mo re efficient when the charged region A was present in the mutant (1-334). T he N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also ab le to interact with ER, pointing to the monomeric N-terminal adenosine trip hosphate binding region of Hsp9O (1-224) as the region necessary and suffic ient for interaction. These results contribute to understand the Hsp90-depe ndent process responsible for conformational competence of ER.