The non-activated glucocorticoid receptor: structure and activation.
Glucocorticoid hormone receptors are present in the soluble fraction of target cell homogenates as large entities (Mr approximately 300,000) that are unable to interact with DNA. These large complexes contain an Mr approximately 94,000 steroid- and DNA-binding polypeptide, in association with an Mr approximately 90,000 non-ligand-binding entity, which has been identified as a heat shock protein, hsp90. This protein has been purified to near homogeneity as a component of the non-activated receptor complex. Characterization of the purified protein revealed its presence as a dimer in the large receptor form. Dissociation of the receptor-hsp90 complex can be induced by heat treatment only when ligand is bound to the receptor, as demonstrated by specific DNA-binding assay and sucrose gradient ultracentrifugation, hsp90 represents ca 1% of total proteins in rat liver cytosol, and milligram amounts were purified using a combination of high performance ion exchange and gel permeation chromatography. Monospecific antibodies were raised in rabbits. They were found to precipitate the intact non-activated glucocorticoid receptor, as well as the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment, indicating that hsp90 interacts with the steroid-binding domain of the glucocorticoid receptor. Finally, translation of glucocorticoid receptor mRNA in reticulocyte lysate yields a protein which also interacts with hsp90 and binds to DNA only after ligand-binding and heat treatment. Thus, the glucocorticoid receptor is synthesized in a non-activated form also in vitro.
Gustafsson
J A
JA
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
Wikström
A C
AC
Denis
M
M
D000818:Animals; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008954:Models, Biological; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D004247:DNA