Comparison of corticosteroid binder IB with the α-chymotrypsin- and RNase-treated hepatic glucocorticoid receptors

Abstract

Rat liver and kidney cytosolic extracts contain the glucocorticoid receptor (binder II) and corticosteroid binder IB, both of which possess the steroid- and DNA-binding domains. Since it has been speculated that the smaller binder IB may be generated from binder II by proteolysis, the chymotrypsinproduced receptor fragment in rat liver cytosol has been compared with binder IB in terms of charge, size and DNA binding characteristics. The [ 3H]triamcinolone acetonide-receptor complex is converted to a smaller fragment by short term digestion (10°C, 30 min) with 100 μg/ml α-chymotrypsin. Although the chymotrypsin fragment produced from previously heat-activated binder II and binder IB both exhibit DNA-binding capability, they differ in charge and size. Whereas the α-chymotrypsin-treated receptor has a Stokes radius of 30 Å and elutes from DEAE-cellulose at 0.06 M potassium phosphate in a linear salt gradient, binder IB has a Stokes radius of 20 Å and elutes in the buffer wash of the DEAE-cellulose column. Thus, while binder IB can be resolved from the heat-activated form of the [ 3H]TA-receptor on DEAE, the heat activated α-chymotrypsin product elutes from the anion exchange resin at the same ionic strength as intact activated binder II (i.e. at 0.05 M potassium phosphate), and the unactivated intact receptor elutes at about 0.20 M potassium phosphate. A more extended digestion with α-chymotrypsin (24 h, 0°C) results in elimination of the DNA binding site without further reduction of the Stokes radius or change in the elution pattern from DEAE-cellulose. Furthermore, molybdate completely blocks formation of binder IB but does not inhibit the production of the receptor fragment by α-chymotrypsin. Treatment of the hepatic [ 3H]TA-receptor complex with RNase has no effect on the charge, size or DNA binding properties of the bound receptor. These results suggest that RNase does not activate the [ 3H]TA-receptor complex nor does it produce a IB-like component in the liver cytosol. The present results are consistent with the hypothesis that binder IB is formed in vitro by a process which may not involve proteolytic cleavage or RNase-induced modification of the glucocorticoid receptor (binder II).