A panel of hybridomas producing monoclonal antibodies to the rat glucocorticoid receptor has been produced, and four stable clones from that panel have already been established. Two of the stable clones, BuGR1 and BuGR2, produce antibodies recognizing different epitopes on the receptor. BuGR1 antibody has been successfully used by us to perform immunoaffinity chromatography of the glucocorticoid receptor and by a collaborator to identify an expression vector containing a recombinant clone of rat cDNA which produces a peptide containing the BuGR1 epitope. We propose to: 1) develop stable hybridoma clones producing antibodies to epitopes located in the steroid-binding and DNA-binding regions of the receptor; 2) purify the activated and native forms of the receptor by conventional and immunoaffinity techniques, and; 3) determine the subunit, multimeric composition of the receptor. Individual hybridoma clones will be screened for their ability to detect the meroreceptor, for their ability to block steroid binding to the receptor, and for their ability to block receptor binding to DNA. To purify the activated and native forms of the receptor, DNA cellulose chromatography (for the activated receptor) or steroid-affinity chromatography (for the native receptor) will be followed by immunoaffinity chromatography. A peptide containing the antibody epitope will be used to competitively elute the receptor from the immunoaffinity column in an undenatured form. The multimeric structure of the immunopurified, denatured receptor will be studied by classical bifunctional cross-linking methods. Ultimately, the fruits of this project will be a detailed description of the structural features of the rat glucocorticoid receptor, including its amino acid sequence, and an understanding of the relationship of those features to biologically important functions of the glucocorticoid receptor.
| van der Weijden Benjamin, W S; Hendry 3rd, W J; Harrison 3rd, R W (1990) The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated in vivo. Biochem Biophys Res Commun 166:931-6 |
