Binding of in vitro-translated, ³⁵S-labeled GR to liganded myGR immunoprecipitated from whole-cell extracts is dependent on the receptor hinge region. Immunoprecipitates from whole-cell extracts prepared from Sf7 cells expressing myGR and treated with 10⁻⁶ M Dex for 1 h prior to harvesting [Sf7 (GR⁺), lanes 5 to 8 and 18 to 22] or control Dex-treated cells lacking myGR (Sf7, lanes 9 to 12 and 23 to 27) were tested for binding to in vitro-translated GR derivatives (lanes 5 to 12 and 18 to 27), whose composition is summarized schematically at the top of each panel. In vitro-translated WT GR and GR peptides X795, 505C, and 547C were treated with 10⁻⁶ M Dex, while GR peptide XΔ509–631 was treated with 0.4 M NaCl to strip away the chaperone complex prior to incubation with liganded, immunoprecipitated myGR. Dissociation of the in vitro-translated GRs from the hsp complex on Dex and NaCl treatment was confirmed by sedimentation analysis of the receptor over 15 to 30% sucrose gradients (Savory et al., unpublished). Lanes 1 to 4 and 13 to 17 show 10% of the input from the in vitro translations.

Amino acids 505 to 550 are sufficient for GR-GR binding in vitro. (A) GST-GR_X550 containing a protein kinase A (PKA) recognition site purified free of the GST moiety and labeled with ³²P by protein kinase A was tested for binding to GST-GR fusion proteins. The compositions of all of the proteins used in the assays are summarized at the top. GR_X550 binding is compared to 10% of the input peptide shown in lane 1 and was resolved by autoradiography of an SDS-PAGE gel (18% polyacrylamide). (B). Tricine-Tris PAGE (16.5% polyacrylamide) of ³²P-labeled GR_505–550 (lanes 1 to 4) and the analogous peptide from the monomeric nuclear receptor FTZ-F1 from Drosophila (lanes 5 to 8) following binding reactions performed in increasing concentrations of the glutaraldehyde cross-linking agent as indicated. The arrow indicates the position of migration of the cross-linked GR peptides.

Binding of in vitro-translated ³⁵S-labeled GR to myGR immunoprecipitated from whole-cell extracts requires amino acids 511 to 539. Immunoprecipitates from whole-cell extracts prepared from Sf7 cells expressing myGR [Sf7 (GR⁺); lanes 4 to 6] or control cells (Sf7, lanes 7 to 9) as indicated were tested for binding to the in vitro-translated GRs, whose composition is illustrated schematically at the top. All samples were treated with 10⁻⁶ M Dex. GR binding is compared to 10% of the input from the in vitro translations shown in lanes 1 to 3.

Solution oligomerization of GR in mammalian cells is independent of the targeting of GR to DNA. (A) The new constructs (GFP-GR_R496H, buGR_505C, and myGR_505CNl1⁻) used in this experiment are summarized schematically. A schema of the myGR_NL1⁻ construct is shown in Fig 7. (B) In situ immunofluorescence analysis of the localization of GFP-GR_R496H, myGR_NL1⁻, buGR_505C, and myGR_505CNL1⁻ expressed singly and in the combinations shown. Dex treatment at 10⁻⁶ M for 1 h was performed as indicated. Specific localization of the GR constructs was visualized as follows: GFP-GR_R496H using direct fluorescence; myGR_NL1⁻ by indirect immunofluorescence analysis using antibody 9E10 followed by a rhodamine-red-conjugated anti-mouse secondary antibody (allowing the detection of myGR_NL1⁻ in the presence of GFP-GR_R496H); buGR_505C by indirect immunofluorescence using antibody BuGR₂ and a fluorescein-conju- gated anti-mouse secondary antibody; myGR_505CNL1⁻ by indirect immunofluorescence using antibody 9E10 followed by a fluorescein-conjugated anti-mouse secondary antibody. Photomicrographs of the immunofluorescence pattern of representative cells are shown to the left, while quantification of observations of a minimum of 150 cells for each sample in each of a minimum of three independent experiments performed in triplicate is shown to the right. The localization of myGR_NL1⁻ prior to steroid treatment is shown in Fig. 7 and is not repeated here. GR localization in each cell was categorized as completely or mostly nuclear (solid grey bars), equally distributed throughout the cell (stippled bars), or localized predominantly or exclusively to the cytoplasm (white bars). The error bars indicate the standard errors of the means. Bar, 10 μm.

MR-dependent nuclear uptake of GR is mediated through the GR LBD. In situ immunofluorescence analysis of the ability of full-length GR and MR (buMR) to influence the subcellular localization of GFP-GR_N524NL1⁻ and myGR_540C (summarized schematically at the top) in COS7 cells is shown. Cells examined in the absence of Dex treatment are indicated (− Dex), while all other samples were examined following a 1-h treatment with 10⁻⁶ M Dex (− Dex). The receptor combinations expressed are as indicated in the middle of each data set. The localization of GR_WT and buMR_WT prior to hormone treatment was shown previously and is not repeated here. Localization of GFPGR_{N524 NL1⁻} was determined by observation of direct fluorescence from fixed cells, while localization of GR and buMR was done by indirect immunofluorescence using antibody buGR₂ and localization of myGR_540C was done by indirect immunofluorescence using antibody 9E10. Quantification of our observations, performed as described in the legend to Fig. 7, is displayed to the right, while representative photomicrographs are shown to the left. MR-GR localization in each cell was categorized as completely or mostly nuclear (solid gray bars), equally distributed throughout the cell (stippled bars), or localized predominantly or exclusively to the cytoplasm (white bars). Bar, 10 μm.

Binding of in vitro-translated, ³⁵S-labeled GR to myGR immunoprecipitated from whole-cell extracts is dependent on hsp dissociation. (A) Immunoprecipitates with myc epitope antibody 9E10 from whole-cell extracts prepared from Sf7 cells expressing myGR (lanes 4 to 6) or control cells lacking GR (lanes 7 to 9) were tested for binding to in vitro-translated firefly luciferase (Luc.) or WT GR. Both the cells and in vitro-translated receptors were treated with 10⁻⁶ M Dex, as indicated in the figure and described in detail in Materials and Methods. For binding reactions performed in the absence of steroid, association of GR with the chaperone complex was stabilized in cell extracts prior to immunoprecipitation and in the binding assays through the inclusion of 20 mM Na₂MoO₄ in all buffers as indicated. Specific myGR binding (Bound, lanes 4 to 9) was revealed by SDS-PAGE and fluorography and is compared to 10% of the in vitro-translated proteins added to the binding-assay mixture (Input, lanes 1 to 3). Loading of the GR immunoprecipitates with or without hormone is revealed by Western blotting in lanes 10 and 11. (B) 9E10 immunoprecipitates from Sf7 and Sf7 (GR⁺) cells were tested for binding to in vitro-translated GR. In lanes 1 to 3, the cells and in vitro-translated receptors were treated with 10⁻⁶ M Dex, while in lanes 4 to 6, the immunoprecipitates and in vitro-translated GRs were treated with 0.4 M NaCl to strip the hsp-immunophilin complex from the receptor prior to binding. Binding of in vitro-translated GR is compared to 10% of the input from the in vitro translation. Loading of the GR immunoprecipitates in the binding assay is revealed by Western blot analysis in lanes 7 and 8.

Amino acids 505 to 568 are required for GR-GR binding in a GST pulldown assay. Binding of in vitro-translated, ³⁵S-labeled, Dex-treated, WT GR to GST-GR fusion proteins (B), whose composition is summarized schematically in the top panel A, is compared to 10% of the input ³⁵S-labeled GR from the in vitro translation (lane 1). A Coomassie blue-stained SDS-PAGE gel of the loading of the GST fusion proteins on the Sepharose beads is shown in the middle panel.

Amino acids 505 to 556 are required for GR-GR binding in a yeast two-hybrid assay. Relative activation of a β-galactosidase reporter gene from Gal4 response elements on coexpression of the indicated Gal-GR fusion proteins, whose composition is summarized at the top, following treatment of liquid cultures for 16 h with 10⁻⁶ M DAC or vehicle. The error bars indicate the standard errors of the means from three independent experiments performed in duplicate. All Gal-GR fusion proteins were expressed to similar levels as determined by Western blot analysis of yeast extracts (Savory et al., unpublished).

Amino acids 511 to 539 are required for oligomerization of GR in mammalian cells in vivo. Physical interaction between GRs in transiently transfected COS7 cell was assessed by the ability of WT GR to promote the nuclear localization of myGR derivatives lacking the major GR NLS, NL1. (A) Western analysis of whole-cell extracts prepared from COS7 cells expressing WT GR and myGR derivatives singly (lanes 1 to 3) and in combination (lanes 4 and 5) with the common buGR antibody illustrating the minimum 4:1 ratio of expression for WT GR to myGR derivatives used in the immunofluorescence assays in panel B. A summary of the composition of the constructs employed in this experiment is given at the top. (B) In situ immunofluorescence analysis of the localization of WT GR using antibody buGR (GR WT) and of the myGR derivatives using anti-myc antibody 9E10 (remaining panels) before Dex treatment (− Dex) and following a 1-h treatment with 10⁻⁶ M Dex (+ Dex). Photomicrographs of the immunofluorescence pattern of representative cells are shown to the left, while quantification of observations of a minimum of 150 cells for each sample in each of a minimum of three independent experiments performed in triplicate are shown to the right. As described previously (48), GR localization in each cell was categorized as completely or mostly nuclear (solid grey bars), equally distributed throughout the cell (stippled bars), or localized predominantly or exclusively to the cytoplasm (white bars). The error bars indicate the standard errors of the means. Bar, 10 μm.

MR promotes the nuclear uptake of GR irrespective of GR amino acids 511 to 539. (A). Western blot of COS7 cell extracts comparing the levels of the MR and GR constructs in panel B, using the antibody BuGR. A schematic of the buMR construct is shown at the top of the panel. The GR constructs used are summarized in Fig. 7. (B) In situ immunofluorescence analysis of GR and MR peptide localization in transfected COS7 cells prior to Dex treatment (− Dex) or following 1 h treatment with 10⁻⁶ M Dex (− Dex). The localization of the GR constructs prior to hormone treatment is shown in Fig. 7 and is not repeated here. Antibody buGR was used to identify the localization of buMR, while myc epitope antibody 9E10 was used to localize the myGR derivatives in both the absence and presence of buMR. Photomicrographs of the immunofluorescence pattern of representative cells are shown to the left, while quantification of our observations, performed as described in the legend to Fig. 7 is displayed to the right. MR-GR localization in each cell was categorized as completely or mostly nuclear (solid grey bars), equally distributed throughout the cell (stippled bars), or localized predominantly or exclusively to the cytoplasm (white bars). Bar, 10 μm.