Control of glucocorticoid and progesterone receptor subcellular localization by the ligand-binding domain is mediated by distinct interactions with tetratricopeptide repeat proteins

Abstract

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 Flag-FKBP52 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.

Figure 1

Western Blot Profile of TPR proteins in L929, WCL2 and COS-1 Cells. A, Expression levels of GR, FKBP52, FKBP51, Cyp40, and PP5 were measured by Western-blotting of whole-cell lysates from L929, WCL2 and COS-1 cells. Tubulin was blotted as a loading control. B, Quantitation of TPR levels. Densitometric values were normalized as percent of WCL2 and represent the means ± SEM of three independent experiments.

Figure 2

Comparison of TPR Intracellular Distribution. Indirect immunofluorescence was performed in COS-1 and wild-type (WT) mouse embryonic fibroblast (MEF) cells using antibodies against FKBP52, PP5 and Cyp40. Results are representative of three independent experiments with at least 100 cells inspected per condition.

Figure 3

Comparison of Hormone-free GR in L929 and WCL2 Cells by Fluorescence and Fractionation. A, GR localization in L929 and WCL2 cells was detected by indirect immunofluorescence using FiGR monoclonal antibody against receptor, as described under Materials and Methods. Cells were treated with vehicle or dexamethasone (DEX, 1 µM for 1 h), as indicated. B, L929 and WCL2 cells treated with or without dexamethasone (DEX, 1 µM, 1 h) were fractionated into cytosolic (C) and nuclear (N) extracts. Immunoadsorption was performed using FiGR antibody against GR, followed by Western blotting. Results in B are representative of three independent experiments.

Figure 4

GR Complexes in WCL2 Cells Recruit Higher Levels of FKBP52 and Lower Amounts of FKBP51 Compared to L929 Cells. A, Analysis of TPR content in GR heterocomplexes from L929 and WCL2 cells. Aliquots of L929 and WCL2 cytosol were immunoadsorbed with FiGR antibody against GR or non-immune mouse IgG (NI). Samples were split and analyzed by Western blotting with antibodies against GR, HSP90, FKBP52, FKBP51, Cyp40, and PP5. B, Quantitation of TPR protein levels in GR heterocomplexes was accomplished by densitometric scanning of the films, followed by subtraction of non-immune values and normalization to amount of GR protein in each condition. Values represent the means ± SEM of three independent experiments. Cyp40 values are omitted due to lack of interaction with either GR.

Figure 5

Over-expression of FKBP51 Alters GR Localization in WCL2 Cells, with No Effect of FKBP52 on L929 Cell GR. A, L929 and WCL2 cells were transiently transfected or not with FLAG-tagged FKBP52 or FLAG-tagged FKBP51, respectively. Whole cell extracts were analyzed for GR, FLAG construct and tubulin (loading control) by Western blotting. B, Analysis of FLAG 51 content in GR complexes from WCL2 cells. WCL2 cells were mock transfected or transfected with FLAG-tagged FKBP51, followed by immunoadsorption of cytosols with FiGR antibody against GR or non-immune mouse IgG (NI). Samples were analyzed by Western blotting with antibody against GR or FLAG epitope. C, Analysis of FLAG 52 content in GR complexes from L929 cells. L929 cells were transfected and analyzed as above, except FLAG-tagged FKBP52 was used. E, Intracellular localization of GR in FLAG-FKBP51 or FLAGF-KBP52 transfected cells. Indirect immunofluorescence of GR in L929 and WCL2 cells following mock or FLAG-FKBP transfection was performed as described under Materials and Methods. Results in A – D are representative of three independent experiments. In Panel E, a minimum cohort of 100 cells per condition was inspected.

Figure 6

Domain Structure of GFP-tagged Wild-type and Chimeric Constructs of GR and PRB. A, Schematic representation of wild-type human GR and human PRB linked to green fluorescent protein (GFP) at the N-terminal domain (NTD). Also shown are chimeric constructs in which the ligand-binding domains (LBD) of GR and PRB are exchanged. Helices 1 – 5 are principal regions of each LBD previously shown by Nordeen to control receptor localization in the absence of hormone (22). These helices coincide with the signal transduction domain (STD) previously shown by Pratt to be a major site for HSP90 interaction (26, 27). DBD, DNA-binding domain; H, Hinge region. B, Western blot profile confirming approximately equal expression of each construct (as numbered in Panel A) in COS-1 cells. Lysates from COS-1 cells 48 h post-transfection were used for Western blot detection of GFP-tagged receptors with antibody against GFP.

Figure 7

Ligand-binding Domains of GR and PRB Control Differential Localization and TPR Specificity. A, LBD controls GR and PRB localization. COS-1 cells were transfected with the indicated wild-type or chimeric receptor expression vectors. Fluorescence microscopy was performed on live, unfixed cells. B. Recruitment of distinct TPRs by GR and PRB is controlled by the LBD. Cytosols were prepared from COS-1 cells transfected with wild-type or chimeric receptor constructs. Equal aliquots of cytosol were used for immunoadsorption with antibody against GFP or non-immune mouse IgG (NI). Samples were split and analyzed by Western blotting with GFP antiserum to detect receptors, or antibodies against FKBP51, FKBP52, PP5 and Cyp40. HC, antibody heavy chain. Results in A and B are representative of three independent experiments. In Panel A, a minimum cohort of 100 cells per condition was inspected.

Figure 8

Characterization of MEF Cells Deficient in FKBP52, FKBP51 or PP5. A, Western blot analysis of TPR proteins in MEF cells from TPR KO mice (see Experimental Procedures). B, GR of WT MEF cells enters into complexes with FKBP51 and PP5. Cytosols from WT MEF cells were immunoadsorbed with FiGR antibody against GR or with non-immune IgG (NI). Samples were split and analyzed by Western blotting for GR, HSP90, FKBP52, FKBP51, Cyp40 and PP5. Results in A and B are representative of five and two independent experiments, respectively.

Figure 9

Analysis of GFP-GR and GFP-PRB by confocal microscopy in WT, FKBP51 KO, FKBP52 KO, and PP5 KO MEF Cells. MEF cells were transfected with GFP-GR or GFP-PRB, followed by 1 h treatment with vehicle (shown) and confocal microscopy on live, unfixed cells. Cells were also treated with 100 nM dexamethasone or 20 nM R5020, as appropriate, for 1 h (data not shown – see text for discussion). In all cases, hormone treatment of receptor in TPR-deficient cells caused nuclear translocation equivalent to that seen in WT cells. A minimum cohort of 100 cells per condition was inspected for each condition.