The cyclosporin A-binding immunophilin CyP-40 and the FK506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor.
We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein (hsp90.hsp70.hsp56) heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. CyP-40 is present in both native glucocorticoid receptor heterocomplexes and receptor heterocomplexes reconstituted with rabbit reticulocyte lysate, and the presence of CyP-40 in the receptor heterocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showing that both immunophilins are in native heterocomplex with hsp90. However, immunoadsorption of hsp56 does not yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not exist in the same heterocomplex with hsp90. Both purified CyP-40 and hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the binding of hsp56, consistent with the presence of a common immunophilin binding site on hsp90. Our data also suggest that there are at least two types of untransformed glucocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 and another that contains CyP-40. The role played by the immunophilins in steroid receptor action is unknown, but it is clear that the peptidylprolyl isomerase activity of immunophilins is not required for glucocorticoid receptor-hsp90 heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate.
Owens-Grillo
J K
JK
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109, USA.
Hoffmann
K
K
Hutchison
K A
KA
Yem
A W
AW
Deibel
M R
MR
Handschumacher
R E
RE
Pratt
W B
WB
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002474:Cell-Free System; D006224:Cricetinae; D016572:Cyclosporine / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D022021:Tacrolimus Binding Proteins; D014162:Transfection
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D016572:Cyclosporine; D022021:Tacrolimus Binding Proteins