The molybdate-stabilized L-cell glucocorticoid receptor isolated by affinity chromatography or with a monoclonal antibody is associated with a 90-92-kDa nonsteroid-binding phosphoprotein.
We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel analysis of the protein A-Sepharose-bound material resulting from incubating the monoclonal antibody with a mixture of 32P-labeled cytosol and [3H]dexamethasone mesylate-labeled cytosol demonstrates identity of the 98-100-kDa [3H]dexamethasone mesylate-labeled band with the 98-100-kDa 32P-labeled band and clear separation from the nonsteroid-binding 90-92-kDa phosphoprotein. The results of immunoblot experiments demonstrate that the 90-92-kDa protein is structurally distinct from the 98-100-kDa steroid-binding protein. As the 90-92-kDa nonsteroid-binding phosphoprotein co-purified with the 98-100-kDa uncleaved form of the glucocorticoid receptor by two independent methods, one of which is based on recognizing a steroid-binding site and the other of which is based on recognizing an antibody binding site, we propose that the 90-92-kDa phosphoprotein is a component of the molybdate-stabilized, untransformed glucocorticoid-receptor complex in L-cell cytosol.
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D003600:Cytosol / Q000032:analysis; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D007739:L Cells / Q000032:analysis*; D051379:Mice; D008961:Models, Structural; D008982:Molybdenum / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives; D014222:Triamcinolone Acetonide / Q000378:metabolism; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D003907:Dexamethasone; D008982:Molybdenum; D010750:Phosphoproteins; D011965:Receptors, Glucocorticoid; D012685:Sepharose; D014222:Triamcinolone Acetonide; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A; C044659:molybdate; C031710:dexamethasone 21-methanesulfonate