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Structure and Function of Cytosolic Glucocorticoid Receptors in WEHI-7 Mouse Thymoma Cells: Receptor Composition and Phosphorylation

  • Dirk B. Mendel
  • Jack E. Bodwell
  • Lynda I. Smith
  • Allan Munck

Summary

The primary goal of these studies was to determine whether the cytosolic glucocorticoid receptor is dephosphorylated during activation in the intact cell. To address this question WEHI-7 mouse thymoma cells were grown with [32P]orthophosphoric acid or [35S]methionine to label the receptor. The nonactivated (non-DNA-binding) and activated (DNA-binding) complexes, formed by incubation of the cells with 200 nM triamcinolone acetonide (TA) at 37 C. were separated on DNA-cellulose. purified using the BuGRl monoclonal antibody, and analyzed by Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). In some experiments. steroid-binding proteins were identified by their specific association with the affinity label [3H]dexamethasone 21-mesylate (DM).

Nonactivated complexes contain a 100 kilodalton (100 kDa) steroid-binding subunit and a 90 kilodalton (90 kDa) non-steroid-binding subunit, both of which are methionine-containing phosphoproteins. Under conditions in which the receptor is saturated with [3H]DM. the 100 kDa subunit has 2–3 moles of phospate for every mole of bound steroid. Following activation, the cytosolic activated complex does not contain the 90 kDa protein. The phosphate content of the 100 kDa steroid-binding-protein is the same for the nonactivated and activated forms of the cytosolic receptor.

From these results we conclude that the nonactivated glucocorticoid-receptor complex is a heteromeric structure which dissociates upon activation, and that activation of the complex in the intact cell does not result in net dephosphorylation of the 100 kDa steroid-binding protein.

Keywords

Glucocorticoid Receptor Phosphate Content Triamcinolone Acetonide Triamcinolone Acetonide Control Monoclonal Antibody 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Martinus Nijhoff Publishing, Boston 1987

Authors and Affiliations

  • Dirk B. Mendel
    • 1
  • Jack E. Bodwell
    • 1
  • Lynda I. Smith
    • 1
  • Allan Munck
    • 1
  1. 1.Department of PhysiologyDartmouth Medical SchoolHanoverUSA

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