Control of Glucocorticoid and Progesterone Receptor Subcellular Localization by the Ligand-Binding Domain Is Mediated by Distinct Interactions with Tetratricopeptide Repeat Proteins<xref ref-type="fn" rid="fn2"></xref>

The TPR proteins FKBP52, FKBP51, Cyp40, and PP5 are found in steroid receptor (SR) complexes, but their receptor-specific preferences and roles remain unresolved. We have undertaken a systematic approach to this problem by examining the contribution of all four TPRs to the localization properties of glucocorticoid (GR) and progesterone (PR) receptors. The GR of L929 cells was found in the cytoplasm in a complex containing PP5 and FKBP51, while the GR of WCL2 cells was nuclear and contained PP5 and FKBP52. Cyp40 did not interact with the GR in either cell line. To test whether FKBP interaction determined localization, we overexpressed Flag-tagged FKBP51 in WCL2 cells and FlagFKBP52 in L929 cells. In WCL2 cells, the GR exhibited a shift to greater cytoplasmic localization that correlated with recruitment of Flag-FKBP51. In contrast, Flag-FKBP52 was not recruited to the GR of L929 cells, and no change in localization was observed, suggesting that both cell-type-specific mechanisms and TPR abundance contribute to the SR-TPR interaction. As a further test, GR-GFP and PR-GFP constructs were expressed in COS cells. The GR-GFP construct localized to the cytoplasm, while the PR-GFP construct was predominantly nuclear. Similar to L929 cells, the GR in COS interacted with PP5 and FKBP51, while PR interacted with FKBP52. Analysis of GR-PR chimeric constructs revealed that the ligand-binding domain of each receptor determines both TPR specificity and localization. Lastly, we analyzed GR and PR localization in cells completely lacking TPR. PR in FKBP52 KO cells showed a complete shift to the cytoplasm, while GR in FKBP51 KO and PP5 KO cells showed a moderate shift to the nucleus, indicating that both TPRs contribute to GR localization. Our results demonstrate that SRs have distinct preferences for TPR proteins, a property that resides in the LBD and which can now explain long-standing differences in receptor subcellular localization. Steroidal control of physiology requires the activation of steroid receptors (SR), which serve as regulators of differential gene expression (1, 2). Prior to hormone binding, all members of the SR family are known to enter into large heteromeric complexes containing the molecular chaperone HSP90 and the cochaperone p23 (3). However, a number of additional cochaperones have been identified that seem to variably interact with SR complexes (4, 5). These are FK506binding protein 52 (FKBP52), the closely related FK506binding protein 51 (FKBP51), cyclosporin A-binding protein (Cyp40), and protein phosphatase 5 (PP5). A common feature of these proteins is the presence of imperfect tetratricopeptide repeat (TPR) motifs that serve as protein-protein interaction domains (6). Indeed, TPR proteins enter into SR complexes by directly binding to HSP90 at its C-terminal TPR acceptor site (7-9). Interestingly, most studies suggest that the TPR acceptor site of HSP90 can accommodate only one TPR protein at a time (10-12). This fact means that several distinct SR heterocomplexes are possible, even in the same cell, based on TPR protein composition. Thus, many reports of interactions of SR with FKBP51, FKBP52, Cyp40, and PP5 have been published, and it has become the conventional wisdom that all SRs can and do interact with all four of these TPRs. According to this school of thought, the four TPRs regulate distinct, but as yet undefined, stages of the signal pathway common to all SRs, yet a competing school of thought exists. It holds that receptors can be preferentially regulated by one TPR over another. In this study, we address these competing models by assessing the contribution of † This study was supported in part by National Institutes of Health Grants DK43867 (E.R.S.), DK70127 (E.R.S. and W.S.), and DK73402 (W.S. and E.R.S.) and the Riley Children’s Foundation (W.S.). * To whom correspondence should be addressed: Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3035 Arlington Ave., Toledo, OH 43614-5804. E-mail: [email protected]. Fax: (419) 383-4182. Phone: (419) 3832871. ‡ University of Toledo College of Medicine. § Indiana University School of Medicine. 1 Abbreviations: SR, steroid receptor; GR, glucocorticoid receptor; PR, progesterone receptor; HSP90, heat shock protein 90; FKBP52, FK506-binding protein 52; FKBP51, FK506-binding protein 51; PP5, protein phosphatase 5; Cyp40, cyclophilin 40; TPR, tetratricopeptide repeat; LBD, ligand-binding domain; NLS, nuclear localization signal; CLS, cellular localization signal. Biochemistry 2008, 47, 10471–1048