Stability and sequence-specific DNA binding of activation-labile mutants of the human glucocorticoid receptor

The stability and DNA-binding properties of activation-labile (act1) human glucocorticoid receptors (hGRs) from the glucocorticoid-resistant mutant 3R7.6TG.4 were investigated. These receptors are able to bind reversibly associating ligands with normal affinity and specificity, but become unstable during attempted activation to the DNA binding form [Harmon et al. (1984) J. Steroid Biochem. 21, 227-236]. Affinity labeling and immunochemical analysis demonstrated that act1 receptors are not preferentially proteolyzed during attempted activation. In addition, analysis of binding to calf thymus DNA showed that after loss of ligand, act1 receptors retain the ability to bind to DNA nonspecifically. A 370 bp MMTV promoter fragment containing multiple GREs and an upstream 342 bp fragment lacking GRE sequences were used to assess the binding of act1 hGR to specific DNA sequences. Immunoadsorption of hGR-DNA complexes after incubation with 32P-end-labeled fragments showed that both normal and act1 hGR bound selectively to the GRE-containing fragment in an activation-dependent manner. Binding of both normal and act1 hGRs could be blocked with a synthetic oligonucleotide containing a perfect palindromic GRE, but not with an oligonucleotide in which the GRE was replaced by an ERE. Analogous results were obtained for normal and act1 hGR activated in the absence of ligand, or after incubation with the glucocorticoid antagonist RU 38486. These results suggest that sequence-specific binding of the hGR does not require the presence of bound ligand and suggest a role for the ligand in trans-activation of hormonally responsive genes.