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

Abstract

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.

Involvement of the glucocorticoid receptor in stress-induced apoptosis of leukemic cells

“…23 DEX also induced rapid nuclear translocation of part of the cytosolic GC receptors, which relocated to the cytosol upon removal of the hormone during the induction phase of about 8-10 h. 19 S49 cells were also sensitive to growth factor deprivation, achieved by growing the cells in medium containing 1% serum, or exposure to cold or heat as described in the next paragraph. Figure 1 illustrates that low serum and heat shock caused nuclear translocation of the GC receptor to a much greater extent than in DEX-treated cells.…”

“…19 Although stress conditions and the drug MIBG could influence this delicate balance at multiple points, it is of note that heat shock proteins are major acceptors of adenosine phosphates, resulting in post-translational mono(ADP-ribosylation). Heat shock or glucose deprivation cause rapid, post-translational decrease of the mono(ADP-ribosylation) of heat shock proteins in various cells 33 and MIBG is a known inhibitor of cellular mono(ADP-ribosylation).…”

“…19 Variant cell lines were generated in a protocol that selected for cells which resisted glucocorticoidmediated cell death but remained sensitive to growth inhibition by the steroid. To this end, cells were grown in 10 −7 M DEX for 72 h, which killed most of the cells (surviving fraction Ϸ 10 −4 ).…”

“…19 In studies involving DEX-exposed cells, the hormone was also added to control cultures immediately before cell harvest by centrifugation. Nuclear translocation of the cytosolic GC receptor was probed in Western blots of 500 mM KCl extractable proteins from nuclear fractions, using methods described previously.…”

“…Nuclear translocation of the cytosolic GC receptor was probed in Western blots of 500 mM KCl extractable proteins from nuclear fractions, using methods described previously. 19 Immunoblotting was performed with monoclonal antibody GR49, kindly donated by Dr Westphal (Philipps Universität, Marburg, Germany), on the protein equivalent of 1 × 10 6 cells or on the equivalent of 1 × 10 7 cells with antiserum MAI-510, clone BuGR2 (Affinity Bioreagents, Neshanic Station, USA). Proteins were visualized with ECL or with 125 I-IgG, followed by quantitation with phosphorimaging.…”