The effect of oxidation/reduction on the charge heterogeneity of the human glucocorticoid receptor.
In order to investigate the molecular basis for the charge heterogeneity which has been reported for the glucocorticoid receptor, we have analyzed the [3H]dexamethasone mesylate ([3H]DM)1 affinity labeled receptor from HeLa S3 cells by high resolution two-dimensional gel analysis. The [3H]DM labeled glucocorticoid receptor from HeLa cells exists as a population of 5-6 isoforms which range over approx. 0.6 pI units but have similar molecular weights. This heterogeneity is apparently the result of modification(s) of a single gene product since the affinity labeled receptor from Chinese hamster ovary (CHO) cells transfected with the human glucocorticoid receptor cDNA displays the same pattern of heterogeneity on two-dimensional gels. Since previous one-dimensional gel studies from our laboratory showed that the structure of the [3H]DM labeled glucocorticoid receptor from HeLa cells is highly susceptible to sulfhydryl group modification, we investigated the potential role of the same modifications in the apparent charge heterogeneity of the glucocorticoid receptor. Treatment of the affinity labeled receptor with iodoacetamide which alkylates free sulfhydryl groups and irreversibly prevents the formation of intra- or intermolecular disulfide bonds, reproducibly resulted in the appearance of 5-6 discrete isoforms of the receptor protein. Treatment with dithiothreitol, a reversible reducing reagent, resulted in detection of 3 to 4 isoforms of the glucocorticoid receptor. In marked contrast, treatment with sodium tetrathionate, which induces intramolecular disulfide bond formation, resulted in only one detectable isoform of the [3H]DM labeled glucocorticoid receptor. These data demonstrate that the oxidation/reduction state of sulfhydryl groups within the receptor protein can account for much of the charge heterogeneity of this ligand dependent transcription factor.
Silva
C M
CM
Department of Biochemistry, University of North Carolina, Chapel Hill 27599.
Cidlowski
J A
JA
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000031:analogs & derivatives* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014316:Tritium
D000345:Affinity Labels; D007461:Iodoacetates; D011965:Receptors, Glucocorticoid; D014316:Tritium; D003907:Dexamethasone; C031710:dexamethasone 21-methanesulfonate; D004229:Dithiothreitol; D019807:Iodoacetic Acid