DISTINCT FUNCTIONS OF THE 90 KDA HEAT-SHOCK PROTEIN (HSP90) IN ESTROGEN AND MINERALOCORTICOSTEROID RECEPTOR ACTIVITY - EFFECTS OF HSP90 DELETION MUTANTS

Recent studies have confirmed that the 90 kDa heat-shock protein (hsp9 0) interacts both in vitro and in vivo with steroid receptors, encoura ging further detailed physicochemical and functional analysis of its c haperone role. Thus, to explore the relationship between hsp90 and rec eptors, the baculovirus system was used to overexpress the chick hsp90 alpha. (chsp90) along with the chick oestradiol receptor (cER) or the human mineralocorticosteroid receptor (hMR). These receptors were abl e to form 9 S complexes with chsp90, demonstrating the association of the co-expressed recombinant proteins. Three mutants of chsp90 (Delta A, Delta B and Delta Z) have been created by deletion of the A (residu es 221-290) and B (530-581) regions, rich in charged amino acids, and the 2 (392-419) region, a putative leucine zipper. After co-expression , anti-receptor antibodies immunoprecipitated the cER hMR complexed wi th the wild-type chsp90, the Delta B or the Delta Z mutant, but not wi th the Delta A chsp90, indicating that deletion of the A region of chs p90 leads to a lack of interaction with these receptors. The hormone b inding capacity of the cER was unaffected after its co-expression with each of the three mutants. In contrast, the hMR co-expressed with the Delta B mutant failed to bind aldosterone, a finding confirmed in viv o by the absence of hormone-induced hMR nuclear translocation. Thus th e B region is required for high-affinity ligand binding by the hMR. Ou r results suggest that the A region (but not the B or Z regions) is in volved in binding of chsp90 to the cER and hMR, while the B region is essential for hormone binding by the hMR, consistent with a chaperone function for hsp90.