title: Optimal ligand binding by the recombinant human glucocorticoid receptor and assembly of the receptor complex with heat shock protein 90 correlate with high intracellular ATP levels in Spodoptera frugiperda cells.
abstract: The full-length human glucocorticoid receptor (hGR), overexpressed in Spodoptera frugiperda (Sf9) cells, associates with heat shock protein 90 (hsp90) and hsp70 and binds dexamethasone with high affinity. Baculovirus infection of Sf9 cells grown in TNM-FH medium results in the rapid depletion of glucose from the medium within 24 h. Noting a discrepancy between hGR protein levels and ligand binding capacity in such cultures, we hypothesized that the depletion of glucose from the medium could result in intracellular ATP depletion and consequently affect the ligand binding capacity of the recombinant hGR. Supplementation of the Sf9 culture medium with additional glucose resulted in a three-fold increase in intracellular ATP levels, and a three-fold increase in 3H-dexamethasone binding capacity, without altering the protein levels of hGR, hsp90 or hsp70. However, more hsp90 co-immunoprecipitated with hGR from cells grown in glucose supplemented medium. Our data support the hypothesis that high-affinity ligand binding by hGR requires the ATP-dependent formation of the hGR:hsp90 heterocomplex. Besides having practical consequences for the production of recombinant GR and other related proteins, our findings could ultimately have relevance in diseases such as diabetes mellitus.
authors.affiliation: Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston 77555-0645, U.S.A. gsriniva@mspo1.med.utmb.edu
authors.forename: G
authors.initials: G
authors.lastname: Srinivasan
authors.forename: J F
authors.initials: JF
authors.lastname: Post
authors.forename: E B
authors.initials: EB
authors.lastname: Thompson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D003907:Dexamethasone / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D011233:Precipitin Tests; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D014316:Tritium
chemical_list: D003470:Culture Media; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D014316:Tritium; D003907:Dexamethasone; D000255:Adenosine Triphosphate; D005947:Glucose