Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related cochaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well de®ned, and so we examined in vivo the in¯uences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding af®nity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the Nand C-terminal ends, respectively. Both microdomains are characterized by ␣-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways.The immunophilin components of the unactivated estrogen receptor, cyclophilin 40 (CyP-40), 1 and FKBP52, share significant sequence homology in their C-terminal regions (1) and represent separate classes of peptidylprolyl cis-trans-isomerases with binding specificities for the immunosuppressants cyclosporin A and FK506, respectively (2). These immunophilins display a similar structural organization of their functional domains characterized by an N-terminal region with overlapping isomerase and ligand binding domains and a conserved C-terminal segment that incorporates a 3-unit tetratricopeptide repeat (TPR) domain terminated by a potential site for calmodulin binding (1). We have previously speculated that the TPR domain may mediate the protein interaction properties of . This is consistent with evidence that similar repeat units in members of the TPR gene family are involved in functional association with target proteins (3).FKBP52 binds hsp90 within steroid receptor complexes (4) and also exists in association with hsp90 in the absence of receptor (5, 6). The interaction of FKBP52 with hsp90 has been studied extensively (7), and there is recent evidence that the TPR domain, localized in the C-terminal region of FKBP52, is fundamentally important for hsp90 binding (8). The structural similarity between CyP-40 and FKBP52 has led several groups to propose that the immunophilins may have a similar or perhaps competing role in cellular function (8 -10). In this regard, a recent report describes the association of human CyP-40 with hsp90 and provides evidence that the C-terminal, FKBP52-like domain determines this interaction ...

The tetratricopeptide repeat (TPR) motif is one of many repeat motifs that form structural domains in proteins that can act as interaction scaffolds in the formation of multi-protein complexes involved in numerous cellular processes such as transcription, the cell cycle, protein translocation, protein degradation and host defence against invading pathogens. The crystal structures of many TPR domain-containing proteins have been determined, showing TPR motifs as two anti-parallel α-helices packed in tandem arrays to form a structure with an amphipathic groove which can bind a target peptide. This is however not the only mode of target recognition by TPR domains, with short amino acid insertions and alternative TPR motif conformations also shown to contribute to protein interactions, highlighting diversity in TPR domains and the versatility of this structure in mediating biological events.