Kevin A. Hutchison

A Model of Protein Targeting Mediated by Immunophilins and Other Proteins That Bind to hsp90 via Tetratricopeptide Repeat Domains

We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR). FKBP52/hsp56 binds to hsp90 via its tetratricopeptide repeat (TPR) domains, it is not required for GR·hsp90 heterocomplex assembly, and it is thought to play a role in targeted movement of the GR. In this work we examine the hsp90 binding of four proteins (FKBP52/hsp56, CyP-40, p50, Mas70p) thought to be involved in targeted protein trafficking. FKBP52/hsp56 and CyP-40 (each with three TPRs), localize to the nucleus and nucleoli, respectively, and form relatively weak complexes with hsp90 that are competed by a CyP-40 fragment containing its three TPRs. The p50 component of the Src·hsp90 and Raf·hsp90 heterocomplexes localizes to cytoskeletal fibers extending from the perinuclear region to the plasma membrane and forming a rim under the plasma membrane of endothelial cells. p50, Mas70p (seven TPRs), which is a receptor for mitochondrial import, and the p60 (six to eight TPRs) component of the steroid receptor·hsp90 heterocomplex assembly system bind very tightly to hsp90 in a manner that is not competed by the CyP-40 fragment. However, bacterially expressed p60 blocks the binding of p50, Mas70p, FKBP52/hsp56, and CyP-40 to purified hsp90. The data are consistent with binding of all of these proteins to a site on hsp90 that is a general TPR domain acceptor. Our localization and binding data are used to develop a model in which proteins that are chaperoned by hsp90 move as dynamic complexes to their cellular sites of action, with the TPR-containing protein participating in targeting the movement of the complexes.

Regulation of Glucocorticoid Receptor Function through Assembly of a Receptor‐Heat Shock Protein Complexa

Incubation of immunopurified, hormone-free mouse glucocorticoid receptors with rabbit reticulocyte lysate results in ATP-dependent and monovalent cation-dependent assembly of the GR into a heterocomplex with hsp90, hsp70, and hsp56. Heterocomplex assembly is accompanied by conversion of the receptor from a form that does not bind steroid to a high affinity steroid-binding conformation. Reticulocyte lysate also promotes ATP-dependent dissociation of unliganded receptors from a prebound receptor-DNA complex. Receptor released from DNA has been reconstituted into the heat shock protein heterocomplex and converted to the non-DNA-binding state. The reticulocyte lysate also reconstitutes pp60v-src into a heterocomplex containing hsp90 and p50, both of which are components of the native heterocomplex form of the tyrosine kinase in cytoplasm. Although the c-Raf-1 serine/threonine kinase has never been found in native association with hsp90, it can be assembled into a heat shock protein heterocomplex by the ATP-dependent system in reticulocyte lysate.

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 accommodates 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.

Steroid receptor folding by heat-shock proteins and composition of the receptor heterocomplex

Over the past 2 years, reports from several laboratories have supported the proposal that the steroid receptors are bound through the hormone-binding domain to a protein complex that contains three heat-shock proteins-hsp90, hsp70, and hsp56. This receptor-heat-shock-protein heterocomplex accounts for the behavior of the classic 9 S, non-DNA-binding form of the adrenocorticoid, sex hormone, and dioxin receptors. The receptor heterocomplex has now been reconstituted by an enzymatic system in reticulocyte lysate. This represents the first in vitro system for reversing receptor transformation, and this ability to reconstitute the receptor heterocomplex promises rapid advances in our understanding of how these receptors are folded, transported, and regulated by hormone in the cell.

Ability of various members of the hsp70 family of chaperones to promote assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90

To be in a conformation that binds steroid, the hormone-binding domain of the glucocorticoid receptor (GR) must be bound to the 90 kDa heat shock protein (hsp90). Rabbit reticulocyte lysate contains a protein chaperone system that assembles the receptor into a heterocomplex with hsp90 and converts it from a non-steroid-binding to a steroid-binding form. Assembly of the GR-hsp90 heterocomplex requires hsp70, and in this work we examine the activities of four members of the hsp70 protein family in GR-hsp90 heterocomplex assembly. Rabbit reticulocyte lysate was depleted of hsp70 by passing it through a column of ATP agarose, resulting in the inactivation of its GR-hsp90 heterocomplex assembly activity. Addition of purified animal (mouse) or plant (wheat germ) hsp70 to the hsp70-depleted lysate permits assembly of a GR-hsp90 heterocomplex with a high affinity steroid binding site. However, purified hsp70 homologues from bacteria (DnaK) or the endoplasmic reticulum (BiP) do not promote heterocomplex formation, despite the fact that both DnaK and BiP bind to the GR in the assay system. When added to whole (i.e. hsp70-containing) reticulocyte lysate, DnaK and BiP inhibit GR-hsp90 heterocomplex assembly. Wheat germ lysate forms a heterocomplex between mouse GR and plant hsp90, but the addition of purified rabbit hsp70 to the wheat germ lysate does not increase the amount of receptor-wheat hsp90 complex produced, despite the fact that the rabbit hsp70 binds to the GR when it is added to the wheat chaperone system. The conclusion is that binding of hsp70 to receptors does not necessarily reflect a physiologically meaningful interaction. When native receptor heterocomplexes isolated from cytosols contain hsp70, it is likely that the hsp70-bound receptors represent a minority of receptors that have not yet proceeded fully through the receptor heterocomplex assembly process, which includes the dissociation of hsp70 after the binding of hsp90.

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

Abstract 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 32 P-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 32 P-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 [ 32 P]orthophosphate.

High levels of non-activated receptors in glucocorticoid-sensitive S49wt mouse lymphoma cells incubated with dexamethasone.

Upon agonist binding the heteromeric glucocorticoid receptor complex undergoes a conformational change (receptor activation). This event involves the dissociation of a dimer of 90 kDa heat shock proteins. Whereas receptor activation in cytosolic assays is both rapid and irreversible, less is known about the receptor activation and translocation in intact cells during challenge with an agonist. In this paper we report on the receptor status of glucocorticoid-sensitive murine S49 lymphoma cells during dexamethasone exposure. By three different assays, ligand (re)binding, nuclear translocation and hsp90 co-immunoprecipitation, it was found that the majority of the glucocorticoid receptor protein was in a non-activated conformation. Furthermore, prolonged exposure to dexamethasone did not result in increased levels of activated receptors. By assessing receptor activation in situ we found that physiological temperature was less effective in dissociating hsp90 compared to room temperature. These findings indicate that the physiological temperature negatively controls receptor activation, probably due to a thermolabile interaction between the hormone and its cognate receptor.