Glucocorticoid receptor binding to calf thymus DNA. 2. Role of a DNA-binding activity factor in receptor heterogeneity and a multistep mechanism of receptor activation.
In the preceding paper [Cavanaugh, A. H., & Simons, S. S., Jr. (1990) Biochemistry (preceding paper in this issue)], we characterized an apparently identical factor in the cytosol and the nuclear extract of HTC cells that is required for the DNA binding of approximately 43% of the activated receptor-glucocorticoid complexes. In the present study, both those activated complexes that are influenced by this factor and the role of this factor in the process of activation are examined. We find that sodium arsenite inhibits only the DNA binding of those complexes that require factor. Conversely, methyl methane-thiolsulfonate inhibits the DNA binding of only those complexes that are independent of factor. These results provide direct chemical evidence for two populations of activated complexes. Double-reciprocal plots revealed that the increase in DNA binding with endogenous factor occurred by recruiting new complexes for DNA binding as opposed to increasing the binding affinity of existing complexes. These results further suggest that factor associates only with the receptor-steroid complex and does not additionally interact with DNA. A saturable association of factor with complexes was indicated since the amount of available factor in cytosolic solutions decreased after activation of the complexes. Sodium molybdate is known to inhibit the activation of HTC cell receptor-steroid complexes. When factor was added to complexes that had been subjected to activating conditions in the presence of the inhibitor sodium molybdate, no increased DNA binding was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Cavanaugh
A H
AH
Laboratory of Analytical Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Simons
S S
SS
D000818:Animals; D001151:Arsenic / Q000494:pharmacology; D018053:Arsenites; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D003911:Dextrans; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008982:Molybdenum / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D017670:Sodium Compounds; D013950:Thymus Gland / Q000378:metabolism*
D018053:Arsenites; D004268:DNA-Binding Proteins; D003911:Dextrans; D011965:Receptors, Glucocorticoid; D017670:Sodium Compounds; C014674:methyl methanethiosulfonate; C017947:sodium arsenite; D008982:Molybdenum; D004247:DNA; C025614:sephadex; C024687:sodium molybdate(VI); D008741:Methyl Methanesulfonate; D001151:Arsenic