Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains.

Abstract

The glucocorticoid-receptor complex can be subdivided into 3 separate domains by limited proteolysis with trypsin or .alpha.-chymotrypsin. The following characteristics can be separated: steroid-binding activity (domain A), DNA-binding activity (domain B), and immunoactivity (domain C). The separation of the steroid-binding domain from the DNA-binding domain by limited proteolysis of the receptor with trypsin was previously reported. The detection by immunochemical analysis of a 3rd domain of the glucucorticoid receptor, which does not bind hormone, is reported. Immunoactivity was detected by using specific antiglucocorticoid receptor antibodies raised in rabbits against purified rat liver glucocorticoid receptor and the assay used was an enzyme-linked immunosorbent assay. After digestion with .alpha.-chymotrypsin, the immunoactive region of the receptor (domain C) was separated from the other 2 domains (A and B). The immunoactive fragment had a Stokes radius of 2.6 nm. Further digestion with .alpha.-chymotrypsin resulted in separation of the immunoactive fragment to give a fragment having a Stokes radius of 1.4 nm. The immunoactive domain could be separated from the half of the glucocorticoid receptor containing the steroid-binding and the DNA-binding domains (Stokes radius, 3.3 nm) by limited proteolysis of the receptor by .alpha.-chymotrypsin followed by gel filtration or chromatography on DNA-cellulose.