Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506-or cyclosporin Abinding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315-32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormonefree cells. Approximately one-half of the GR⅐hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and ϳ35% contains PP5. Only a small (but undetermined) fraction of the native GR⅐hsp90 heterocomplexes contain the cyclosporin Abinding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [ 3 H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.In cytosols prepared from hormone-free cells, steroid receptors exist in multiprotein complexes that contain hsp90 1 and some hsp90-associated proteins, including p23 and some high molecular weight immunophilins (for review see Refs. 1 and 2). The immunophilins are ubiquitous and conserved proteins that bind immunosuppressant drugs, such as FK506 and cyclosporin A (for review see Ref.3). All members of the immunophilin family have peptidylprolyl isomerase (PPIase) activity, and there are two classes: the FKBPs that bind compounds like FK506 and rapamycin and the cyclophilins (CyPs) that bind cyclosporin A. The drugs bind to the isomerase site on the immunophilin and inhibit cis-trans isomerization in vitro (4).The low molecular weight immunophilins, such as FKBP12 and CyP-18, are thought to be the cellular components responsible for the immunosuppression and are the most studied. Three high molecular weight immunophilins, FK...

The sequential binding of heat shock protein 90 (hsp90) to a series of tetratricopeptide repeat (TPR) proteins is critical to its function as a molecular chaperone. We have used site-directed mutagenesis to clarify the structural basis for the binding of hsp90 to the TPR domain of phosphoprotein phosphatase 5 (PP5). This TPR domain was chosen for study because its threedimensional structure is known. We examined co-immunoprecipitation of hsp90 with wild type and mutant TPR constructs from transfected cells. Only mutations located on one face of the TPR domain affected hsp90 binding. This allowed the identification of a binding groove. Three basic residues that are highly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, while mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutation of Lys-32, another conserved basic residue in the binding groove, also blocked hsp90 binding. The TPR domain of PP5 bound specifically to a 12-kDa C-terminal fragment of hsp90. This binding was reduced by mutation of acidic residues in the hsp90 fragment. These data suggest conservation, among hsp90-binding TPR proteins, of a binding groove containing basic residues that interact with acidic residues near the C terminus of hsp90.