The role of DNA-binding and phosphorylation on nucleocytoplasmic trafficking of the glucocorticoid receptor.

Abstract

To characterize the nuclear transfer defect in the mouse S49 lymphoma, I have used the Arg484-His and Tyr770-Asn single amino acid substituted glucocorticoid receptor (GR) mutants to examine the effects of four factors on nucleocytoplasmic (NC) transport (NCT): defective hormone binding, defective DNA binding, the GR antagonist RU486, and modulators of protein kinase and protein phosphatase activities. A quantitative indirect immunofluorescence assay was developed and used during G$\sb0$ to measure kinetics of nuclear import, cytoplasmic redistribution of nuclear receptors after hormone withdrawal and nuclear re-uptake of cytoplasmic redistributed receptors. I showed that the Tyr770-Asn GR mutant, defective in hormone binding, has no influence on nucleocytoplasmic transport. Kinetic studies with the Arg484-His and other DNA binding GR mutants demonstrated that the DNA binding function of the receptor is an important determinant for the persistence of GR in the nucleus following hormone treatment and withdrawal. I showed that detection of GR in the cytoplasm following hormone withdrawal occurs concomitantly with reformation of the 8-9S complex, a process we termed re-cycling. RU486, a potent GR antagonist (previously shown to block hyperphosphorylation of the receptor) potentiated apparent nuclear retention through an apparent block in receptor re-cycling. Further, this effect could be completely reproduced during withdrawal from agonist by treatment with protein kinase inhibitor, 1-(5-isoquinolinyl-sulphonyl)-2-methylpiperazine (H-7). Conversely, phorbol ester treatment markedly enhanced the initial return of agonist treated nuclear receptors, but not RU486 treated GR, to the cytoplasm. These effects were independent of DNA binding of the receptor. This suggested that a 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-sensitive kinase activity is required for the re-cycling of GR following dissociation of hormone. In energy depletion experiments, using RU486- and H-7 treated cells, I showed that unliganded nuclear GR retained the ability to shuttle between nucleus and cytoplasm. Thus my results are consistent with reincorporation of the receptor into an inactive 8-9S heat shock proteins-complex (re-cycling), but not exit from the nucleus, being blocked by these two agents. Kinetic measurements of loss of nuclear receptors to the cytoplasm after hormone withdrawal, using combined modulators such as forskolin/(TPA) or forskolin/okadaic acid (OA) demonstrated a cancellation effect on apparent nuclear retention of WT receptor. However, the Arg484-His GR mutant in both cases demonstrated either the effects of TPA alone or the forskolin alone respectively. These preliminary data provide evidence of different phosphorylation events playing an important role in DNA binding and subsequent transcriptional activity of GR.