Evidence for microheterogeneity in the structure of human glucocorticoid receptors.
The human glucocorticoid receptor has been selectively affinity labeled with [3H]dexamethasone mesylate by utilizing whole cells to form complexes of steroids and receptors. The nonspecific interaction of [3H]dexamethasone mesylate [[3H]9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione-21-mesylate ([3H]DM)] with proteins containing sulfhydryl groups has been minimized by incubation of intact HeLa S3 cells with 1 X 10(-8) M [3H]DM at 0 C before preparation of cytosol fractions. Under these conditions, which result in the labeling of 30% of the total cellular receptor, [3H]DM binds to a single protein that has a mol wt of about 88,000. Four lines of evidence suggest that this protein, with a molecular mass of approximately 88,000 represents the glucocorticoid receptor. 1) [3H]DM binding is saturable. 2) Glucocorticoids and progesterone, but not estradiol or testosterone, compete with [3H]DM for binding with the 88,000-dalton protein. 3) The sedimentation behavior of the glucocorticoid receptors is quite similar whether they are labeled with [3H]DM or with [3H]dexamethasone. Protein labeled with [3H]DM sediments as an approximately 7.5S species in 5-20% sucrose gradients. Increasing the ionic strength of the buffer during centrifugation produces a receptor form that sediments as a species at about 4.5S. 4) The affinity labeled glucocorticoid receptors display isoelectric focusing patterns nearly identical to those observed for receptors labeled with [3H] dexamethasone. [3H]DM-receptor complexes have been subjected to high resolution two-dimensional gel analysis. The data revealed that the human glucocorticoid receptor consists of a family of at least five proteins with molecular masses of approximately 88,000 which have discrete isoelectric points ranging from 6.5-7.5. We hypothesize that this heterogeneous population of proteins represents multiple species of steroid receptor proteins within the same cell or perhaps post-transcriptional modification of a single protein.
D000345:Affinity Labels / Q000378:metabolism; D002474:Cell-Free System; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000378:metabolism; D007526:Isoelectric Point; D008970:Molecular Weight; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000032:analysis*; D011987:Receptors, Steroid / Q000032:analysis*
D000345:Affinity Labels; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone; C031710:dexamethasone 21-methanesulfonate