Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49 ++ lymphoma cells: Biochemical characterization and interaction with hsp 70 and hsp 90

Charles E. Powell, Cheryl S. Watson, Bahiru Gametchu

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

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 0.6 M 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.

Original languageEnglish (US)
Pages (from-to)271-280
Number of pages10
JournalEndocrine
Volume10
Issue number3
StatePublished - 1999

Keywords

  • Epitope elution
  • Heat shock proteins
  • Membrane glucocorticoid receptor

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

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