Selective deletions in the 90 kDa heat shock protein (hsp90) impede hetero-oligomeric complex formation with the glucocorticosteroid receptor (GR) or hormone binding by GR.
We have developed an in vivo system using coexpression of human glucocorticosteroid receptor (hGR) and chick hsp90 alpha (chsp90) in recombinant virus-infected Sf9 cells to study the formation of hetero-oligomeric complexes. We detected, in the cytosol, hGR complexes containing chsp90 as shown by the displacement of the [3H]triamcinolone acetonide bound hGR "8S" peak on glycerol/sucrose gradients by specific antibodies directed against chsp90 (BF4 and D7 alpha). We took advantage of this system and of the immunoadsorption of hGR containing complexes with anti-hGR antibody to analyze the effect of deletions introduced into the hsp90 molecule on the formation of complexes with the hGR. Deletion of the hydrophilic region "A", between amino-acids 221 and 290, abolished the formation of hGR/chsp90 complexes. Deletion of the hydrophilic region "B" (between amino-acids 530 and 581) or deletion of a leucine repeat region "Z" in the middle of the molecule (amino-acids 392 to 419) still allowed formation of hetero-oligomeric complexes detected by immunoadsorption but the hGR complexes formed with mutated chsp90s were devoid of steroid binding properties. These results are consistent with (1) the possible involvement of the "A" region in the interaction of hsp90 with steroid receptors and (2) a role of B and Z regions in the hsp90 structure for maintaining the steroid binding property of the hGR.
Cadepond
F
F
INSERM U33, Laboratoire Hormones, Le Kremlin-Bicêtre, France.
Jibard
N
N
Binart
N
N
Schweizer-Groyer
G
G
Segard-Maurel
I
I
Baulieu
E E
EE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D009036:Moths; D016297:Mutagenesis, Site-Directed; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014222:Triamcinolone Acetonide / Q000378:metabolism
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D014222:Triamcinolone Acetonide