A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accomodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30180/1/0000565.pd

Glucocorticoid receptor phosphorylation in mouse L-cells

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25[deg]C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37[deg]C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26917/1/0000483.pd

Point Mutations in the 90-kDa Heat Shock Protein Binding Region of the Glucocorticoid Receptor Affect the Functional Characteristics of the Receptor a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72162/1/j.1749-6632.1995.tb31403.x.pd

Localization of the ~12 kDa Mr discrepancy in gel migration of the mouse glucocorticoid receptor to the major phosphorylated cyanogen bromide fragment in the transactivating domain

The intact wild-type mouse glucocorticoid receptor has a theoretical molecular weight of ~86 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of ~98 kDa. It is not known where the unusual primary structure or covalent modification responsible for this anomalous migration is located within the amino acid chain. In the course of examining the pattern of fragmentation of 32P-labeled glucocorticoid receptors from Chinese hamster ovary (CHO) cells containing amplified mouse receptor cDNA, we have found a localized region in the amino-terminal half of the receptor that accounts for this anomalous behavior. Cyanogen bromide treatment of the intact receptor produces a 23.4 kDa (theoretical) fragment consisting of residues 108-324 and containing all of the identified phosphorylated serines within the receptor. We find that the only large resolvable 32P-labeled receptor fragment produced after complete cyanogen bromide cleavage of intact receptors migrates with an apparent molecular weight of ~35 kDa. Because the apparent difference between the theoretical and the experimentally observed molecular weights of this cyanogen bromide fragment is essentially the same as the difference between the theoretical and experimental molecular weights of the intact mouse glucocorticoid receptor, we propose that some feature lying within this fragment accounts for slower migration. Although the existence of an additional phosphorylation site lying within the 15 kDa tryptic receptor fragment containing the DNA-binding domain has been contested, we also demonstrate that this fragment of the mouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing [32P]orthophosphate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30424/1/0000045.pd