IN-VIVO EVIDENCE FOR THE GENERATION OF A GLUCOCORTICOID RECEPTOR HEAT-SHOCK PROTEIN-90 COMPLEX INCAPABLE OF BINDING HORMONE BY THE CALMODULIN ANTAGONIST PHENOXYBENZAMINE

The glucocorticoid receptor (GR) is a ligand-regulated transcription f actor whose ability to bind hormone is thought to be dependent on asso ciation with the 90-kDa heat shock protein (hsp90). In the present stu dy, we have generated a novel form of the GR, in which the receptor re mains complexed to hsp90 but has lost its ability to bind hormone, by treatment of intact cells with the calmodulin (CaM) antagonist phenoxy benzamine (POBA). Treatment of these cells, mouse L929 cells stably tr ansfected with the mouse mammary tumor virus-chloramphenicol acetyltra nsferase (MMTV-CAT) reporter construct, with increasing concentrations of POBA resulted in a concentration-dependent inhibition of dexametha sone (Dex)-induced CAT gene expression, with 100 mu M POBA resulting i n approximately 80% inhibition. This inhibitory effect of POBA was mar kedly reduced ii POBA was added after a short incubation with Dex, sug gesting that the primary effect of POBA was on hormone-induced transfo rmation of the GR. Using a subcellular fractionation technique, POBA i nhibition of CAT gene expression was found to correlate with an inhibi tion of Dex-induced GR nuclear translocation. However, inhibition of t ranslocation was not the primary effect of POBA on the GR signal pathw ay, as POBA was found to reduce GR hormone-binding capacity after trea tment of intact cells. The inhibitory effect of POBA on hormone-bindin g function correlated closely with the inhibitory effect of this drug on CAT gene expression and was not due to an oxidation of sulfhydryl g roups, a condition known to reduce GR hormone-binding capacity. Incuba tion of cytosols from untreated cells with POBA did not decrease GR st eroid-binding capacity, demonstrating that this inhibitory effect was not the result of a competitive antagonism at the ligand-binding site. Quantitation of GR protein in the cytosols of POBA-treated cells reve aled that the decrease in steroid-binding function was not due to a lo ss of GR protein. Surprisingly, the amount of OR-bound hsp90 was also unaltered in response to POBA. Taken together, the above observations provide evidence for a novel state of the GR within intact cells in wh ich hsp90 interaction is but one step in the generation or maintenance of hormone-competent receptors. In addition, these results point to t he potential use of POBA, and possibly other CaM inhibitors, as antago nists of steroid receptor actions.