Abstract
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.
Highlights
From the :j:Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, the Irnepartment of Biochemistry, The Upjohn Company, Kalamazoo, Michigan 49001, and the 'fIDepartment of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90
We show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class
Summary
20479-20484, 1995 Printed in U.S.A. The Cyclosporin A-binding Immunophilin CyP-40 and the FK506-binding Immunophilin hsp Bind to a Common Site on hsp and Exist in Independent Cytosolic Heterocomplexes with the Untransformed Glucocorticoid Receptor*. We have recently shown that hsp, 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 hsp We show that both untransformed glucocorticoid receptor and hsp heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. 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-hsp heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate. Our data suggest that there are at least two types of untransformed steroid receptor heterocomplexes, one that contains hsp and another that contains CyP-40
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