Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments.
The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.
D000345:Affinity Labels / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014357:Trypsin
D000345:Affinity Labels; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D014357:Trypsin; C031710:dexamethasone 21-methanesulfonate