Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49++ lymphoma cells: biochemical characterization and interaction with Hsp 70 and Hsp 90.
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 microM 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 Bmax of 384 fmol/mg protein for mGR, compared to a k(d) of 19.5 nM and a Bmax 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.
Powell
C E
CE
Department of Pediatrics, Medical College of Wisconsin, Milwaukee 53226, USA.
Watson
C S
CS
Gametchu
B
B
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D003907:Dexamethasone / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D007163:Immunosorbent Techniques; D047029:Lactams, Macrocyclic; D008223:Lymphoma / Q000737:chemistry*; D051379:Mice; D015735:Mifepristone / Q000378:metabolism; D010449:Peptide Mapping; D011809:Quinones / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism; D014222:Triamcinolone Acetonide / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D000450:Aldosterone; D003907:Dexamethasone; D014222:Triamcinolone Acetonide; C001277:geldanamycin