New site-directed polyclonal antibody maps N-terminus of occluded region of the non-transformed glucocorticoid receptor oligomer to within bugr epitope

Abstract

Using a 17-mer synthetic peptide for immunization, a polyclonal antibody (WS933) directed against amino acid residues 395–411 of the mouse glucocorticoid receptor (GCR) has been raised and used to probe the significance of this region in forming the receptor oligomer and to localize the truncation site of the mutant GCR of the P1798 lymphosarcoma. This region of the receptor, which encompasses the BUGR epitope, is amino-terminal of and immediately adjacent to the DNA-binding domain. The polyclonal antibody WS933 reacted with both native and denatured forms of the wild-type mouse GCR as judged by its ability to shift the transformed receptor peak on Sephacryl S300 columns, to immunoadsorb the receptor to protein A Sepharose, and by immunoblot analysis where it identified the 98 kDa receptor protein in the cortisol-sensitive line of the P1798 mouse lymphosarcoma. WS933 also reacted with rat and rabbit GCR, but not human GCR. These characteristics were shared by the BUGR-2 monoclonal antibody. Unexpectedly, there were two highly significant differences between WS933 and BUGR-2. The first was the ability of WS933 to bind to the mutant 45 kDa GCR of the cortisol-resistant P1798 lymphosarcoma as judged by its capability of shifting the receptor peak on Sephacryl S300 columns. BUGR-2, in contrast, was unable to shift this mutant receptor peak. Secondly, WS933 was unable to react with the non-DNA-binding form of the wild-type (or mutant) GCR, whereas BUGR-2 could react with the non-DNA-binding form of the wild-type GCR. The first observation suggests that the truncation site of the mutant receptor may lie within a portion of the BUGR domain. Additionally, the second observation implies that at least part of the region lying within amino acid residues 395–411 of the mouse GCR is occluded in the receptor oligomer and that this site only becomes available upon transformation of the GCR to the DNA-binding form. This data provides the first mapping of the amino-terminus of the occluded region of the non-transformed receptor, and suggests that WS933 will be a useful probe for characterizing mutant as well as wild type glucocorticoid receptors.