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

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    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

    Fingerprint

    Glucocorticoid Receptors
    Lymphoma
    Membranes
    HSP90 Heat-Shock Proteins
    Epitopes
    Triamcinolone Acetonide
    Mifepristone
    Peptides
    Proteins
    T-Cell Lymphoma
    Aldosterone
    Sepharose
    Dexamethasone
    Glucocorticoids
    Membrane Proteins
    Salts
    Steroids
    Monoclonal Antibodies
    Cell Membrane

    Keywords

    • Epitope elution
    • Heat shock proteins
    • Membrane glucocorticoid receptor

    ASJC Scopus subject areas

    • Endocrinology

    Cite this

    Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49 ++ lymphoma cells : Biochemical characterization and interaction with hsp 70 and hsp 90. / Powell, Charles E.; Watson, Cheryl S.; Gametchu, Bahiru.

    In: Endocrine, Vol. 10, No. 3, 1999, p. 271-280.

    Research output: Contribution to journalArticle

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