Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49++ lymphoma cells

The membrane glucocorticoid receptor (mGR), previously correlated with glucocorticoid-induced lymphocytolytic competency, was purified under nondenaturing conditions from mGR-enriched mouse S-49 T lymphoma cells. Proteins were immunoaffinity batch adsorbed to BUGR-2 monoclonal antibody-coupled protein A Sepharose 4B beads, and elution by epitope competition was compared with standard denaturation procedures. Elution with BUGR-2 epitope peptides released multiple mGRs (42–150 kDa) and heat shock proteins 70 and 90, suggesting that mGR interacts with these protein chaperones under physiological conditions. The mGR-heat shock protein 90 interaction was inhibited by 1 μM geldanamycin. Several other mGR binding partners were captured and most were dissociated from mGR by 0.6M salt. Peptide maps of purified mGR displayed immunoreactive bands unique to mGR. Scatchard analysis estimated a k_d value of 239 nM and a B_max of 384 fmol/mg protein for mGR, compared to a k_d of 19.5 nM and a B_max of 90.3 fmol/mg protein for the intracellular GR (iGR). The rank order of affinities for mGR were RU-486 > dexamethasone > triamcinolone acetonide=aldosterone. Other steroids had no significant binding affinity. These results show that epitope-purified mGR on the plasma membrane of mouse lymphoma cells is similar but not identical to iGR.