Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB.
Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002850:Chromatography, Gel; D003600:Cytosol / Q000032:analysis; D003636:DEAE-Cellulose; D004247:DNA; D007668:Kidney / Q000032:analysis*; D008099:Liver / Q000032:analysis; D008297:Male; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D014222:Triamcinolone Acetonide / Q000378:metabolism; D014316:Tritium
D000911:Antibodies, Monoclonal; D011965:Receptors, Glucocorticoid; D014316:Tritium; C044659:molybdate; D008982:Molybdenum; D004247:DNA; D003636:DEAE-Cellulose; D014222:Triamcinolone Acetonide