Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.
Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.
Cadepond
F
F
Institut National de la Santé et de la Recherche Médicale U33, Lab Hormones, Le Kremlin-Bicêtre, France.
Binart
N
N
Chambraud
B
B
Jibard
N
N
Schweizer-Groyer
G
G
Segard-Maurel
I
I
Baulieu
E E
EE
D000818:Animals; D002451:Cell Compartmentation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D004252:DNA Mutational Analysis; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014222:Triamcinolone Acetonide / Q000378:metabolism
D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D014222:Triamcinolone Acetonide