Endocrine Abstracts (2002) 3 P237

Analysis of GR/NFkB interactions in living cells

HJ Garside, A Stevens & DW Ray


Endocrine Sciences Research Group, Stopford Building,University of Manchester, Manchester, UK.


Glucocorticoids (Gc) are highly potent anti-inflammatory agents. They act through the glucocorticoid receptor (GR), a protein capable of multiple protein interactions. The pro-inflammatory cytokines, TNF-a and IL-1 signal, in part, through activation of NF-kB. Nf-kB transactivation is inhibited by activated GR. It has been suggested that the GR, and the p65 (RelA) component of NF-kB form a direct contact independent of Gc ligand, and that Gc binding results in inhibition of p65 transactivation function. We sought evidence of protein-protein interaction between GR and p65 using a mammalian dual hybrid system.

In the absence of ligand there was no induction of reporter gene activity when a Rel homology domain (RHD) protein was tested with a variety of GR constructs fused to VP16, comprising ligand binding domain (LBD), DNA binding domain (DBD), and full length GR (FL). Both RHD, and GR proteins were nuclear. This suggests failure to interact in the absence of Gc. The p65-Gal4 fusion induced a 1300 fold increase in luciferase activity. FLGR resulted in 20% repression with dexamethasone, as did wildtype GR. The DBD/LBD resulted in 40% repression, and the LBD alone failed to repress. When repeated with an NRE-Luc reporter, which interacts with the RHD, rather than Gal4 DBD, the repression caused by the DBD/LBD protein was greater than that achieved by either FLGR or wildtype GR (78%, 40% and 40% respectively) and again the LBD fusion did not repress.

In contrast to previous reports (Nissen and Yamamoto, 2000), we saw no evidence of a ligand-independent interaction between GR and RelA. However the VP16 fusion proteins containing FLGR or the DBD/LDB alone repressed NF-kB transactivation suggesting a ligand-induced interaction and subsequent recruitment of a repressor complex to the p65-GR. The LBD alone however failed to repress, highlighting the importance of the DBD. Therefore we propose that ligand binding to the GR is required for interaction with p65 and the conformation of p65 induced by DNA binding influences repressor recruitment.

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