Abstract
Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.
Highlights
Nuclear hormone receptors are dynamic transcription factors that move rapidly through the nucleus and that, based on the results of heterokaryon fusion assays, are believed to shuttle or exchange rapidly between nucleus and cytoplasm
Liganded glucocorticoid receptor (GR) Is Statically Localized to the Nucleus in Live and Digitonin-permeabilized Cells—To examine the mechanism for nuclear export of GR in situ we developed a fluorescence recovery after photobleaching (FRAP) assay that takes advantage of the significant proportion of cells in many established mammalian tissue culture cell lines that are stably maintained in a multinucleated state (Fig. 1)
As demonstrated by examination of the trafficking of a series of synthetic control proteins tagged with green fluorescent protein (GFP), nucleocytoplasmic protein shuttling in this assay is reflected by the rapid reappearance of fluorescence in the photobleached nucleus of a multinucleated cell (Fig. 1A)
Summary
Plasmids—pGFPGR, pGFP-GRNL1-, pDM128, pRevRex, pRevI B␣1–72, pRSV-gal, and pNESGFPPKNLS are described elsewhere [2, 25,26,27]. pGSTGFPNLS expresses a fusion protein comprised of GST and GFP, with the sequence of the SV40 NLS at the C terminus. Sequencing was performed to ensure that the reading frame of each construct was correct, and Western blotting was performed using the GFP antibody JL8 (Clontech) to verify the expected size of each expression product. Cells were seeded onto 40-mm round coverslips (Bioptechs) and transfected with 1 g of the indicated expression construct using 10 l of LipofectAMINETM (Invitrogen). Following transfection cells were cultured in complete serum overnight and seeded onto 22-mm square coverslips. Cells were plated onto 22-mm square coverslips, treated as described, and fixed with 3% paraformaldehyde in PBS for 30 min at 4 °C, followed by incubation with PBS containing 0.1 M glycine for 10 min at 20 °C. Cells were incubated overnight at 4 °C with primary antibodies to calreticulin (C-17, Santa Cruz Biotechnologies) or calnexin (H-70, Santa Cruz Biotechnologies) diluted 1/150 in PBS containing 5% IgG-free bovine serum albumin. Cells were washed three times in PBS and mounted on microscope slides
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