Comparison of DNA binding properties of activated, covalent and noncovalent glucocorticoid receptor-steroid complexes from HTC cells.

Several differences in the interaction with DNA of noncovalent vs. covalent glucocorticoid receptor-steroid complexes are described. HTC cell glucocorticoid receptors were incubated under cell-free conditions with the potent reversible glucocorticoid dexamethasone and with the irreversible antiglucocorticoid dexamethasone 21-mesylate to yield noncovalent and covalent complexes, respectively. Using DNA immobilized on cellulose, we found that the noncovalent dexamethasone complexes were activated (by dilution in pH 8.8 buffer at 0 degree C) to a DNA binding species 2-fold faster than were covalent dexamethasone 21-mesylate labeled complexes. The affinity of activated, noncovalent dexamethasone complexes for DNA in an equilibrium binding assay was 2-fold higher than that of the activated, covalent dexamethasone 21-mesylate complexes. This conclusion was supported by the observations in a DNA-cellulose pellet assay that covalent receptor-steroid complex binding to DNA was disrupted by lower NaCl concentrations than was noncovalent complex binding. The same studies of DNA binding at various NaCl concentrations failed to provide evidence that glucocorticoid receptor-steroid complex binding to DNA is a multistep process. These quantitative distinctions in the DNA binding properties of covalent and noncovalent receptor-steroid complexes represent the first physicochemical differences between the complexes of antiglucocorticoid and glucocorticoid steroids and may partially account for their divergent biological properties.