Distinct functions of the 90 kDa heat-shock protein (hsp90) in oestrogen and mineralocorticosteroid receptor activity: effects of hsp90 deletion mutants.
Recent studies have confirmed that the 90 kDa heat-shock protein (hsp90) interacts both in vitro and in vivo with steroid receptors, encouraging further detailed physicochemical and functional analysis of its chaperone role. Thus, to explore the relationship between hsp90 and receptors, 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 able 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 (residues 221-290) and B (530-581) regions, rich in charged amino acids, and the Z (392-419) region, a putative leucine zipper. After co-expression, anti-receptor antibodies immunoprecipitated the cER or hMR complexed with the wild-type chsp90, the delta B or the delta Z mutant, but not with the delta A chsp90, indicating that deletion of the A region of chsp90 leads to a lack of interaction with these receptors. The hormone binding 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 vivo by the absence of hormone-induced hMR nuclear translocation. Thus the B region is required for high-affinity ligand binding by the hMR. Our results suggest that the A region (but not the B or Z regions) is involved 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.
D000450:Aldosterone / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D017353:Gene Deletion; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007313:Insecta / Q000821:virology; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011960:Receptors, Estrogen / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D011960:Receptors, Estrogen; D018161:Receptors, Mineralocorticoid; D011994:Recombinant Proteins; D000450:Aldosterone