Localization of the approximately 12 kDa 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 molecular weight of approximately 96 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of approximately 98 kDa. It is not known where the unusual primary structure or covalent modification responsible for this anomalous migration is located within the amino acid chain. In the course of examining the pattern of fragmentation of 32P-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 the receptor that accounts for this anomalous behavior. Cyanogen bromide treatment of the intact receptor produces a 23.4 kDa (theoretical) fragment consisting of residues 108-324 and containing all of the identified phosphorylated serines within the receptor. We find that the only large resolvable 32P-labeled receptor fragment produced after complete cyanogen bromide cleavage of intact receptors migrates with an apparent molecular weight of approximately 35 kDa. Because the apparent difference between the theoretical and the experimentally observed molecular weights of this cyanogen bromide fragment is essentially the same as the difference between the theoretical and experimental molecular weights of the intact mouse glucocorticoid receptor, we propose that some feature lying within this fragment accounts for slower migration. Although the existence of an additional phosphorylation site lying within the 15 kDa tryptic receptor fragment containing the DNA-binding domain has been contested, we also demonstrate that this fragment of the mouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing [32P]orthophosphate.
Hutchison
K A
KA
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
Dalman
F C
FC
Hoeck
W
W
Groner
B
B
Pratt
W B
WB
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003488:Cyanogen Bromide; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015534:Trans-Activators; D014162:Transfection; D014357:Trypsin
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010710:Phosphates; D010748:Phosphopeptides; D011965:Receptors, Glucocorticoid; D015534:Trans-Activators; D014357:Trypsin; D003488:Cyanogen Bromide