title: Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate.
abstract: The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.
authors.affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
authors.forename: V
authors.initials: V
authors.lastname: Daniel
authors.forename: A B
authors.initials: AB
authors.lastname: Maksymowych
authors.forename: E S
authors.initials: ES
authors.lastname: Alnemri
authors.forename: G
authors.initials: G
authors.lastname: Litwack
mesh_terms: D000818:Animals; D002474:Cell-Free System; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000032:analysis / Q000096:biosynthesis*; D012156:Reticulocytes / Q000378:metabolism*
chemical_list: D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid