Direct stoichiometric evidence that the untransformed Mr 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex.
We have used three methods to measure the stoichiometry of the glucocorticoid receptor and the 90-kDa heat shock protein (hsp90) in L-cell glucocorticoid receptor complexes that were purified by immunoadsorption to protein A-Sepharose with an anti-receptor monoclonal antibody, followed by a minimal washing procedure that permits retention of receptor-associated protein. In two of the methods, receptor was quantitated by radioligand binding, and receptor-specific hsp90 was quantitated against a standard curve of purified hsp90, either on Coomassie blue stained SDS gels by laser densitometry or on Western blots by quantitative immunoblotting with 125I-labeled counterantibody. The stoichiometry values obtained by densitometry and immunoblotting are 7 and 6 mol of hsp90/mol of receptor, respectively. In a third method, which detects total receptor protein rather than just steroid-bound receptor, the ratio of hsp90 to receptor was determined by immunopurifying receptor complexes from [35S]methionine-labeled L cells, and the amount of 35S incorporated into receptor and hsp90 was corrected for the established methionine content of the respective proteins. In complexes from L cells which are labeled to steady state (48 h), the ratio of hsp90 to GR is 4:1. When immunoadsorbed receptor complexes are washed extensively with 0.5 M NaCl and 0.4% Triton X-100 in the presence of molybdate, the ratio of hsp90 to GR is 2:1. In addition to hsp90, preparations of [35S]methionine-labeled untransformed receptor complex also contain a 55-kDa protein that the conclusion that the untransformed L-cell glucocorticoid receptor exists in cytosol in a much larger heteromeric complex than considered to date.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
E H
EH
Bresnick
F C
FC
Dalman
W B
WB
Pratt
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism; D007163:Immunosorbent Techniques; D007739:L Cells; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; C044659:molybdate; D008982:Molybdenum; D008715:Methionine