LOCALIZATION OF THE SIMILAR-TO-12KDA M(R) DISCREPANCY IN GEL MIGRATION OF THE MOUSE GLUCOCORTICOID RECEPTOR TO THE MAJOR PHOSPHORYLATED CYANOGEN-BROMIDE FRAGMENT IN THE TRANSACTIVATING DOMAIN

The intact wild-type mouse glucocorticoid receptor has a theoretical m olecular weight of similar to 86 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of similar to 98 kDa. It is not known where the unusual primary structur e or covalent modification responsible for this anomalous migration is located within the amino acid chain. In the course of examining the p attern of fragmentation of P-32-labeled glucocorticoid receptors from Chinese hamster ovary (CHO) cells containing amplified mouse receptor cDNA, we have found a localized region in the amino-terminal half of t he receptor that accounts for this anomalous behavior. Cyanogen bromid e treatment of the intact receptor produces a 23.4 kDa (theoretical) f ragment consisting of residues 108-324 and containing all of the ident ified phosphorylated serines within the receptor. We find that the onl y large resolvable P-32-labeled receptor fragment produced after compl ete cyanogen bromide cleavage of intact receptors migrates with an app arent molecular weight of similar to 35 kDa. Because the apparent diff erence between the theoretical and the experimentally observed molecul ar weights of this cyanogen bromide fragment is essentially the same a s the difference between the theoretical and experimental molecular we ights of the intact mouse glucocorticoid receptor, we propose that som e feature lying within this fragment accounts for slower migration. Al though the existence of an additional phosphorylation site lying withi n the 15 kDa tryptic receptor fragment containing the DNA-binding doma in has been contested, we also demonstrate that this fragment of the m ouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing [P-32]orthophosphate.