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Effect of macromolecular-translocation inhibitor-III on binding of activated glucocorticoid-receptor complex to
1Department of Biochemistry, St Marianna University School of Medicine, Kanagawa.
Abstract:
We previously purified macromolecular-translocation inhibitor-III (MTI-III), which inhibits the binding of the activated glucocorticoid-receptor complex (GR) to nuclei, to homogeneity from rat liver, and we found that the purified MTI-III bound to partially purified activated GR under low salt conditions at slightly acidic pH [Liu, G., Okamoto, K., and Isohashi, F. (1993) Eur. J. Biochem. 218, 679-687]. This was the first direct evidence that the inhibitor acts through a direct interaction with the activated GR. In this study, we examined whether the purified MTI-III could interfere with the binding of GR to a DNA fragment containing a specific glucocorticoid-response element (GRE). Under nearly isotonic salt conditions at neutral pH, the activated GR bound to the GRE but not to nonspecific DNA. Under similar conditions, the activated GR also bound to the purified MTI-III. The resulting GR/MTI-III complex did not bind to the GRE. We also found that addition of MTI-III to the GR/GRE complex resulted in time-dependent disruption of the GR/GRE complex and formation of the GR/MTI-III complex. The half-life of the GR/GRE complex in the presence of MTI-III was about 13 min. These results suggest that MTI-III enhances the release of GR from the GR/GRE complex and immediately forms a stable GR/MTI-III complex.
Insights
Macromolecular-translocation inhibitor-III (MTI-III) directly interacts with the activated glucocorticoid receptor (GR). MTI-III prevents GR binding to DNA and promotes its release from DNA-bound complexes.
Area of Science:
- Molecular biology
- Cellular signaling
Background:
- Macromolecular-translocation inhibitor-III (MTI-III) is a purified inhibitor that affects the activated glucocorticoid receptor (GR) complex.
- Previous studies showed MTI-III binds to activated GR under specific conditions, suggesting direct interaction.
Purpose of the Study:
- To investigate if MTI-III interferes with the binding of activated GR to DNA.
- To determine the effect of MTI-III on the GR/DNA complex.
Main Methods:
- Purified MTI-III and activated GR were used.
- Binding assays were performed using DNA fragments with glucocorticoid-response elements (GREs) and nonspecific DNA under neutral pH and near-isotonic salt conditions.
- Time-dependent disruption of GR/GRE complexes by MTI-III was analyzed.
Main Results:
- Activated GR bound to GRE-containing DNA but not to nonspecific DNA.
- Activated GR also bound to MTI-III, forming a GR/MTI-III complex.
- The GR/MTI-III complex did not bind to GRE.
- MTI-III addition to GR/GRE complexes caused time-dependent disruption and formation of GR/MTI-III complexes, with a half-life of approximately 13 minutes for the GR/GRE complex.
Conclusions:
- MTI-III directly interacts with the activated GR.
- MTI-III enhances the dissociation of GR from DNA.
- MTI-III forms a stable complex with GR, preventing its DNA binding.