Glucocorticoid receptor binding to rat liver nuclei occurs without nuclear transport.
The binding of cell-free activated glucocorticoid receptor-steroid complexes from HTC cells to various preparations of HTC and rat liver nuclei has been examined under conditions that did or did not support the nuclear translocation of macromolecules via nuclear pores. To the best of our knowledge, this is the first such study with functionally active isolated nuclei. Conventionally prepared HTC nuclei were found to be porous, as determined from their inability to exclude the fluorescent macromolecule phycoerythrin (PE) at 4 degrees C. Thus the nuclear binding of activated complexes to these nuclei can not involve nuclear translocation. Further studies, using established conditions with sealed nuclei prepared from rat liver, revealed that nuclear translocation of PE containing a covalently linked, authentic nuclear translocation sequence could be obtained at 22 degrees C, but not at 4 degrees C. However, under the same conditions, activated glucocorticoid complexes displayed equal levels of nuclear binding at both temperatures. We therefore conclude that the current translocation conditions with intact rat liver nuclei are not sufficient to reproduce the nuclear transport of glucocorticoid complexes observed in intact cells. The nuclear binding that was seen with intact rat liver nuclei was not affected by aurintricarboxylic acid, which selectively inhibits protein-nucleic acid interactions. The antibody AP-64, shown to be specific for amino acids 506-514 of the nuclear translocation sequence of the rat glucocorticoid receptor, inhibited the nuclear binding of activated complexes, apparently by blocking receptor access to the nuclear membrane. Collectively, these data argue that activated complex binding to nuclei capable of nuclear translocation involves only an association with nuclear membrane components such as nuclear pores. Thus this system, and these reagents, may be useful in future studies of activated complex binding to nuclear pores.
Miyashita
Y
Y
Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD, USA.
Miller
M
M
Yen
P M
PM
Harmon
J M
JM
Hanover
J A
JA
Simons
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001667:Binding, Competitive; D002457:Cell Extracts / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000502:physiology; D019913:Nuclear Localization Signals / Q000276:immunology / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010799:Phycoerythrin / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002457:Cell Extracts; D000939:Epitopes; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D010799:Phycoerythrin; D001312:Aurintricarboxylic Acid; D003907:Dexamethasone