FIG. 7.
Regulation of RXRα nuclear export by its ligands and homodimerization. (A and B) Analysis of subcellular localization of RXRα/C1 in the absence or presence of retinoids. (A) GFP-RXRα/C1 was transfected into HEK293T cells, which were then treated with the indicated retinoid, stained with Hsp60, and analyzed by confocal microscopy. The inhibitory effect of RXR ligands on the cytoplasmic localization of RXRα/C1 was observed in 80% of transfected cells, whereas >90% of transfected cells failed to respond to RAR ligands. (B) Nuclear and cytoplasmic extracts were also prepared and analyzed for expression of GFP-RXRα/C1 by Western blotting with anti-GFP antibody. One of three similar experiments is shown. (C) RXRα/C1 dimerization status determines its subcellular localization. GFP-RXRα/C1 was transfected into HEK293T cells, which were not treated or treated with 9-cis-RA (10−7 M). Nuclear and cytoplasmic extracts were prepared and analyzed by using nondenaturing PAGE and anti-GFP antibody. The same extracts were analyzed by denaturing PAGE for the expression of PARP and Hsp60 to ensure fraction purity. One of two similar experiments is shown. (D) Confocal microscopy analysis of RXR homodimerization-defective mutants. GFP-RXRα/385, GFP-RXRα/LLL, or GFP-RXRα/C1/C432R was transfected into HEK293T cells. Cells were treated with or without 9-cis-RA (10−7 M) and analyzed by confocal microscopy. Approximately 80% of transfected cells showed nuclear localization of GFP-RXRα, which was slightly increased to 85% after treatment with 9-cis-RA. Cytoplasmic localization of GFP-RXRα/385 (90%), GFP-RXRα/LLL (65%), and GFP-RXRα/C1/C432R (85%) was not affected by 9-cis-RA treatment. One of three similar experiments is shown.