Separation of the hormone- and DNA-binding sites of the hepatic glucocorticoid receptor by means of proteolysis

The dexamethasone · receptor complex in rat liver cytosol has a Stokes radius of 61 Å. It may be converted to smaller complexes with Stokes radii of 36 Å, and 19 Å by proteolytic digestion with trypsin, α-chymotrypsin (that only gives rise to 36-Å complex), papain, or an extract of the 1,000 x g to 10,000 x g pellet of liver homogenate. The 61- and 36-Å complexes have dissociation constants of 6 to 8 x 10^-9 M and 6 to 9 x 10^-9 M, respectively, for dexamethasone. The 19-Å complex is very labile in the absence of bound ligand and therefore its affinity to dexamethasone could not be determined. Both the 61- and 36-Å complexes are bound to nuclei and DNA-cellulose following heat activation in the presence of ligand. The 61-Å complex is eluted from a DNA-cellulose column with a linear KCl gradient at 0.11 to 0.13 M KCl, whereas the 36-Å complex is eluted at 0.15 to 0.20 M KCl. The 19-Å complex binds neither to DNA-cellulose nor to nuclei. When nuclei or DNA-cellulose containing bound dexamethasone · receptor complex were digested with trypsin and subsequently extracted or eluted in low ionic strength, the recovered dexamethasone · receptor complex had a Stokes radius of approximately 19 Å. We conclude that the glucocorticoid receptor in liver cytosol may be divided into three parts separable by proteolytic digestion: (a) the steroid-binding site (retained on the 19-Å and 36-Å receptor fragments); (b) the DNA-binding site (retained on the 36-Å, but absent from the 19-Å receptor fragment); and (c) a region with an as yet unknown function and only present on the 61-Å receptor.