1. Translational Regulatory Mechanisms Generate N-Terminal Glucocorticoid Receptor Isoforms with Unique Transcriptional Target Genes 
Nick Z. Lu, John A. Cidlowski 
Molecular Cell 
Volume 18, Issue 3, Pages (April 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Western Blot Detection of Multiple GR Proteins
(A) The expression pattern of hGRα in COS-1 cells. 20 μg lysates from cells transfected with 200 ng of pTRE-hGRα on 6-well plates treated with either vehicle or dexamethasone (DEX, 10 nM) were resolved and detected by using anti-GR 57.
(B) Detection of hGRα produced in vitro by using SDS-PAGE.
(C) The expression pattern of hGRα in COS-1 cells was determined by using three different anti-GR antibodies. The peptide epitopes are as indicated. Mock-transfected cells were used as controls.
(D) Detection of wild-type (wt) and C-terminal truncated hGRα in COS-1 cells using the anti-GR antibody 57. Abbreviations: τ1, minus DNA (DBD) and ligand binding domains (LBD); τ1-DBD, minus LBD.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 One GR Gene Generates Multiple GR Isoforms
(A) Conservation of AUG codons in the GR N-terminal regions.
(B) N-terminal AUGs are indispensable in the production of GR isoforms. AUGs were replaced with codons for threonine (Yudt and Cidlowski, 2001), alanine (not shown), or isoleucine as indicated. Transfection of COS-1 cells and Western blot procedures were described in Figure 1A and the Experimental Procedures. See text for description. Wt, wild-type hGRα.
(C) Detection of one single GR mRNA species produced by hGRα cDNA using 5′ RLM-RACE. Tobacco acid pyrophosphatase treatment, a step to select intact mRNA, was omitted with control samples (Con).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Translation Regulatory Mechanisms Generate Multiple GR N-Terminal Isoforms
(A) The strength gradient of Kozak contexts in the wt hGRα mRNA.
(B) Ribosomal leaky scanning contributes to the production of the hGRα-B and -C isoforms. Transfection of COS-1 cells and Western blot procedures were described in Figure 1A. Abbreviations: wt, wild-type hGRα; Kozak-wt, optimized Kozak context for AUG1; S85stop, serine85 in hGRα replaced with a stop codon; K315stop, lysine315 replaced with a stop codon; and hGRα-A, all N-terminal AUGs except AUG1 were replaced.
(C) The N-terminal coding sequence (1–1005 bp) of hGRα mRNA facilitates ribosome binding. Each fusion gene (200 ng) was transfected into COS-1 cells in 12-well plates. A reporter assay was performed as described in the Experimental Procedures. The ratio of RNL (renilla luciferase)/LUC (firefly luciferase) activities (± SEM) was used as an index of the translation efficiency of the RNL gene. IRES, internal ribosomal entry sites.
(D) The production of hGRα N-terminal isoforms is 5′ cap dependent. In vitro transcription of the hGRα cDNA yielded a single mRNA species of 2.5 kb as in agarose gel analysis (top). Significantly, when used for in vitro translation (products detected by using Western Blot analysis, bottom), the capped hGRα mRNA (lane 1) yielded higher levels of GR isoforms than the uncapped mRNA (lane 2). In addition, cap decoys (free m7G) reduced the expression of all hGRα isoforms (lane 3).
(E) Ribosomal shunting likely contributes to the expression of the hGRα-C and -D isoforms. The codons immediately downstream of the inserted hairpin structures are as indicated. In-frame insertion of a stable hairpin structure increased the size of the upstream AUG product by 3 kD. 10 μg of lysates of COS-1 cells transfected with 200 ng of each construct was detected by using Western blot analysis with anti-GR antibody 57. See text for description.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Expression Pattern of Endogenous GRα Isoforms
(A) Western blot analysis of the GRα isoforms in representative human cell lines. The blots resolving 50 μg of cell lysates were detected by using multiple GR-selective antibodies 59, 57, and AshGR (see Figure 1).
(B) Western blot analysis of the GRα isoforms in rat tissues. The blot resolving 50 μg of tissue lysates from a representative animal detected by using anti-GR antibody 57 is shown (left). One-way ANOVA and Tukey post-hoc analysis (n = 4, right) were used to compare the relative levels of GRα isoforms among tissues. Densitometry was performed by using NIH Image, and the level of each GR isoform was expressed as a percentage (± SEM) of the total GR level. Significantly different from levels of at least three other tissue types, *p < Significantly different from the level of the GRα-A isoform in the same tissue, +p < 0.05.
(C) Western blot analysis of the GRα isoforms in a series of mouse tissues. The blots using 50 μg of tissue lysates from a representative animal detected with anti-GR antibodies 57 and 59 are shown. Additional bands in between GR-C and GR-D isoforms are not present in all tissues and may be degradation products or tissue-specific isoforms.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Functional Assays for the hGRα N-Terminal Isoforms
(A) Subcellular localization of the hGRα isoforms was analyzed in stable U-2 OS cells by using immunocytochemistry (left two panels) and in COS-1 cells transiently expressing YFP-hGRα isoforms using confocal microscopy (right two panels).
(B) All isoforms were detected at comparable levels by using Western blot analysis in COS-1 cells transiently expressing the hGRα isoforms. 10 μg lysates from cells transfected with 200 ng of each hGRα isoform cDNA on 6-well plates were resolved and detected by using anti-GR antibody 57.
(C) In the same cells, the transactivation activity (± SEM) of each hGRα isoform was increased by DEX in a dose-dependent manner.
(D) EC50 values of the hGRα isoforms in inducing reporter gene expression. RLU, normalized relative light units using GRE2-Luc reporter genes.
(E) Comparable levels of the hGRα N-terminal isoforms in stably expressing U-2 OS cells were detected by using Western blot analysis.
(F) In the same cells, the transactivation activity (± SEM) of each GR isoform was increased by DEX.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Combinatorial Effects of the hGRα N-Terminal Isoforms
(A) In COS-1 cells, 20 ng of the hGRα-A isoform is coexpressed with 2–20 ng of the hGRα-C3 or 2–40 ng of the hGRα-D3 isoform. RLU, normalized relative light units (± SEM) using GRE2-Luc reporter genes.
(B) In U-2 OS cells stably expressing two GR isoforms, the level of the hGRα-A level is constant, and the level of hGRα-C3 or hGRα-D3 isoform can be adjusted by using doxycycline.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Gene-Regulation Profiles of the hGRα Isoforms
(A) Each of the hGRα N-terminal isoforms regulated a unique set of genes. Total numbers of genes induced (red) or repressed (blue) by each isoform are shown. Dotted lines mark the numbers of genes regulated by all isoforms.
(B) Venn diagrams illustrating the number of genes regulated by each hGRα isoform compared to that by the wt hGRα (red). The number in each intersection represents the number of genes commonly regulated by the wt hGRα and one of the isoforms. The other numbers indicate the numbers of unique genes regulated by each individual isoform.
(C) ANOVA analysis revealed that 587 genes were regulated to different degrees by at least two different hGRα isoforms. The numbers of genes regulated to different degrees by each pair of hGRα isoforms are also indicated.
(D) Real-time PCR verification of representative genes. Cells were treated with either vehicle or DEX (100 nM, 3 hr). RNA samples were prepared as described in Microarray Analysis. Primer probe sets and 7900 HT Sequence Detection System were obtained from Applied Biosystems, and all experiments were performed according to the manufacture’s manual. Induced genes are indicated in red, and repressed genes are indicated in green for all hGRα isoforms. Representative genes regulated by a single hGRα isoform are also indicated. See text for description of gene names. Significantly regulated from vehicle treatment (± SEM, Student’s t test, n = 4, *p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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