Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membranebased steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.The glucocorticoid receptor (GR) 1 is a ligand-activated transcription factor whose macromolecular structure in the absence of hormone contains heat shock protein 90 (Hsp90) and tetratricopeptide repeat (TPR) proteins. To date, four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) have been found in so-called "mature" GR complexes, i.e., GR that is competent for binding of hormone. Because Hsp90 contains only one binding site for these proteins, four distinct GR heterocomplexes have been identified on the basis of TPR protein content (reviewed in ref 1). Although such heterogeneity implies differential functions for each GR complex, very little is known about this process. In recent work by our laboratory, differential roles for FKBP52 and FKBP51 in the regulation of hormone-induced transport of GR were uncovered (2). It was found that binding of...

We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR⅐Hsp90⅐FKBP52⅐dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNAbinding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport. The glucocorticoid receptor (GR)1 is a hormone-activated transcription factor that requires hormonally driven movement to its site of action within the nucleus. In the absence of hormone, the GR is recovered in the cytosolic fraction of cells as an oligomeric complex containing one molecule of receptor and two molecules of heat shock protein 90 (Hsp90), to which the receptor binds directly (for review see Ref. 1). An intriguing recent development, however, is that hormone-free receptor is not found as a single, well defined complex but exists as a mixture of complexes. Although all of these complexes contain receptor and Hsp90, each contains only one molecule of either FKBP52, FKBP51, Cyp40, or PP5. The latter proteins have been classified as TPR domain proteins based on the presence of several tetratricopeptide repeat domains that are their sites of interaction with Hsp90 (2, 3). FKBP52, FKBP51, and Cyp40 are also members of the immunophilin family of proteins (4 -6). Thus, it is now clear that up to four distinct receptor heterocomplexes are possible, even within the same cell or tissue; yet almost nothing is known about the differential roles served by each immunophilin in steroid receptor responses.It is generally accepted that the first event in hormonal activation of GR is dissociation of hormone-bound GR from Hsp90 and the TPR proteins, followed by nuclear translocation of the GR and all other downstream events. Hormone-induced dissociation of the complex is a rapid event, occurring both in the intact cell (7) and in cytosolic preparations (8). In cytosols, dissociation of GR complexes has been shown to require warming (typically 20 -25°C) in addition to hormone, and this process can be blocked by molybdate and other transition metal oxyanions (8, 9). Indeed, because of the ability of molybdate to effectively block dissociation, it has been assumed that molybdate-stabilized receptors are more or less "frozen" in their native, untransformed state even when GR is bound with hormone. In this work, we have examined this assumption by measuring the effect of hormone on the immunophilin content of GR hetero...

The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug FK506. FKBP52 exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity, which is inhibited by the binding of FK506--properties that it shares with the smaller but better-studied immunophilin, FKBP12. Unlike FKBP12, however, FKBP52 does not mediate the immunosuppressive actions of FK506 and, due to its larger size, contains additional numerous functional domains. One such structure is a series of tetratricopeptide repeat (TPR) domains, which serve as binding sites for the ubiquitous and abundant molecular chaperone, Hsp90. It is this property as a TPR protein that best characterizes the known cellular roles of FKBP52. Here, we review the structural features of FKBP52 and relate them to the evolving and diverse functions of this protein. Although the most recognized role of FKBP52 is in regulation of steroid receptor signaling, other less well-known functions are also discussed.