Use of high-resolution two-dimensional gel electrophoresis and affinity labeling to probe glucocorticoid receptor structure and function.
The possible role of posttranslational modification in glucocorticoid receptor regulation was investigated. Glucocorticoid receptor (GR), prepared from the human B-cell line IM-9 and affinity labeled with [3H]-dexamethasone 21-mesylate, was examined by a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution two-dimensional gel electrophoresis. Two-dimensional electrophoresis of immunopurified [3H]dexamethasone 21-mesylate-labeled GR revealed the presence of two isoelectric species (apparent pI approximately to 5.7, and 6.0-6.5). Both forms were present in preparations of unactivated receptor. After GR activation, the pI of neither isoform was altered, indicating that activation does not involve covalent charge modification of the steroid-binding protein. However, only the pI 6.0-6.5 isoform bound to DNA, suggesting that covalent charge modification of the GR can alter its ability to bind to DNA. Two-dimensional electrophoresis of tryptic and chymotryptic fragments showed that the charge heterogeneity responsible for the two GR isoforms is located in a Mr 26,500 tryptic fragment derived from the steroid-binding domain of the protein. In addition, analysis of [3H]dexamethasone 21-mesylate-labeled tryptic fragments suggests that the Mr 26,500 fragment corresponds to residues 499-743 of the human GR. These results demonstrate that posttranslational modification of the steroid-binding domain may regulate the ability of the protein to bind to DNA.
Harmon
J M
JM
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Smith
A C
AC
Elsasser
M S
MS
D000345:Affinity Labels; D000595:Amino Acid Sequence; D003907:Dexamethasone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000502:physiology
D000345:Affinity Labels; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone