Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein.
The glucocorticoid receptor is present in cytosol prepared from cell extracts of nonhormone-treated cells as a large nonactivated (i.e. non-DNA binding) 9 S heteromeric complex which contains the Mr approximately 90,000 heat shock protein, hsp90. hsp90 is expressed under physiological conditions in mammalian cells and is also present in reticulocyte lysate, as assessed by Western immunoblotting using specific anti-hsp90 antibodies. We have translated glucocorticoid receptor mRNA in reticulocyte lysates. The receptor synthesized under cell-free conditions also interacts with hsp90 both in the presence and absence of ligand, as determined by sucrose gradient centrifugation. The in vitro synthesized glucocorticoid receptor does not bind to DNA-cellulose but can be converted to a DNA binding form following labeling with dexamethasone and heat treatment. Thus, the glucocorticoid receptor is synthesized in a nonactivated form under cell-free conditions. These data indicate that the 9 S glucocorticoid receptor complex found in cytosol does not represent an artifact due to cell homogenization and supports the existence in vivo of the glucocorticoid receptor-hsp90 complex.
Denis
M
M
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
Gustafsson
J A
JA
D000818:Animals; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D006360:Heat-Shock Proteins / Q000502:physiology*; D046911:Macromolecular Substances; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis*; D012156:Reticulocytes / Q000378:metabolism
D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid