Assessment of glucocorticoid receptor-heat shock protein 90 interactions in vivo during nucleocytoplasmic trafficking.
Unliganded glucocorticoid receptors (GRs) that reside in the cytoplasm exist as heteromeric complexes comprised minimally of 90-kDa heat shock protein (hsp90) hsp70 and p56, a 56-kDa immunophilin. The binding of hsp90 to the GR occurs primarily through its carboxy-terminal, ligand-binding domain. Dissociation of GR-associated proteins accompanies hormone binding and leads to the exposure of its various functional domains. Although an association with hsp90 presumably masks the GR nuclear localization signal sequence, the recent demonstration of the coimport of GR and hsp90 into nuclei has led to the hypothesis that hsp90 facilitates GR interactions with the nuclear transport machinery. In this report we examined whether the dynamics of GR/hsp90 interactions in vivo influences its trafficking both into and out of the nucleus. GR/hsp90 complexes were stabilized in vivo by the introduction of sodium molybdate to cultured cells using a liposome-mediated delivery system. In agreement with previous in vitro studies, we found that stabilization of GR/hsp90 complexes in live cells severely restricts hormone-dependent nuclear import of GR. Constitutive nuclear import of a GR deletion derivative that does not bind hsp90 is unaffected by intracellular administration of molybdate, demonstrating that the inhibitory effects of molybdate require the coupling of the nuclear localization signal sequence to the GR ligand-binding domain. Interestingly, molybdate treatment traps both GR and progesterone receptor in the cytoplasm of cells chronically exposed to hormone, indicating that shuttling GRs and progesterone receptors can export, but not reimport into nuclei in the presence of molybdate. This result implies that the reassociation of recycled receptors with hsp90 must be an obligatory step for receptors that exit the nucleus to reacquire the capacity for nuclear import.
Yang
J
J
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
DeFranco
D B
DB
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D000882:Haplorhini; D007668:Kidney; D008114:Liver Neoplasms, Experimental; D008982:Molybdenum / Q000494:pharmacology; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D008982:Molybdenum; C024687:sodium molybdate(VI)