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

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.

Different Regions of the Immunophilin FKBP52 Determine Its Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic Dynein

“…2). Of these two immunophilins, CyP-40 is known to be more weakly bound in GR⅐hsp90 heterocomplexes (12), and less is associated with the washed hsp90 immune pellet (Fig. 2).…”

“…It is clear that TPR proteins bind to hsp90 with different affinities, ranging from the loosely bound CyP-40, which can be washed off hsp90 immune pellets with low salt buffers (12), to the very tightly bound Hop, some of which binds to hsp90 despite the presence of extremely high levels of competing TPR domain fragments of CyP-40 (20) or PP5 (33). In Fig.…”

“…In addition to hsp90, native steroid receptor heterocomplexes contain one of several proteins that possess tetratricopeptide repeats (TPRs), which are degenerative sequences of 34 amino acids involved in protein-protein interactions (3). The TPR proteins in steroid receptor⅐hsp90 heterocomplexes include high molecular mass immunophilins such as FKBP52 (4 -7), FKBP51 (8 -10), and CyP-40 (11)(12)(13) as well as the protein serine/threonine phosphatase PP5 (14,15). The TPR domains of these proteins are required for their binding to hsp90 (14, 16 -19), and the TPR proteins and protein fragments containing only the TPR domains compete with each other for binding (12,15,18,20) to a TPR domain acceptor site (20) that is located in the C-terminal 12-kDa domain of hsp90 (21).…”

“…The TPR proteins in steroid receptor⅐hsp90 heterocomplexes include high molecular mass immunophilins such as FKBP52 (4 -7), FKBP51 (8 -10), and CyP-40 (11)(12)(13) as well as the protein serine/threonine phosphatase PP5 (14,15). The TPR domains of these proteins are required for their binding to hsp90 (14, 16 -19), and the TPR proteins and protein fragments containing only the TPR domains compete with each other for binding (12,15,18,20) to a TPR domain acceptor site (20) that is located in the C-terminal 12-kDa domain of hsp90 (21). The immunophilins have been shown to exist in independent receptor⅐hsp90⅐FKBP52 and receptor⅐hsp90⅐CyP-40 heterocomplexes (12,13), and inasmuch as the TPR proteins compete for the binding of each other, these complexes must be very dynamic in the sense that a single receptor⅐hsp90 heterocomplex could be associated sequentially with several different TPR proteins over a short time.…”

Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90

“…Several proteins involved in the hsp90 chaperone pathway possess structurally related tetratricopeptide repeat motifs that mediated their interaction with hsp90 and͞or hsp70 (8)(9)(10)(11). The common tetratricopeptide repeat acceptor site for steroid receptorassociated immunophilins and Hop is located in the dimerization domain of hsp90 near the C terminus (see Fig.…”